Carbanion in Water

Acc. Chem. Res.
2001, 34, 981-988
Formation and Stability of Carbocations and Carbanions in Water and Intrinsic Barriers to Their Reactions
JOHN P. RICHARD,* TINA L. AMYES, AND MARIA M. TOTEVA
Department of Chemistry, University at Buffalo, State University of New York, Buffalo, New York 14260-3000 Received July 26, 2001 ABSTRACT
chemical reactivity is of interest to both organic chemists and biochemists.
Lifetimes in water as short as 10-11 s have been determined for carbocations and carbanions by referencing the rate of their reaction with solvent species to that for the appropriate clock reaction, and equilibrium constants have been determined as the ratio of rate constants for their formation and breakdown. Rateequilibrium correlations for these organic ions are often poor and sometimes even defy the simple generalization that reactivity increases with decreasing stability. This seemingly confusing body of data can be understood through consideration of the both the Marcus intrinsic barrier and the thermodynamic driving force to reaction of these organic ions.
Only a few laboratories are investigating the reactions of carbocations and carbanions in aqueous solution. The restricted interest in these important reactions has provided motivation for the following work described in this Account. (1) Development of experimental methods to obtain reliable estimates of lifetimes, down to the picosecond regime, of carbocations and carbanions in water, which are intermediates of chemical and biochemical reactions. (2) Characterization of the dynamics of carbocation-anion ion pair intermediates of solvolysis reactions. (3) Determination of equilibrium constants for formation of carbocations and carbanions. (4) Rationalization of the changes in these rate and equilibrium constants that occur with variations in substrate structure.
Reaction Clocks
Many sophisticated studies of reactive intermediates have focused on determination of absolute rate constants by directly monitoring the disappearance of the intermediate generated by laser flash photolysis.2 We have used simpler, but effective, indirect methods which reference the unknown rate constant for reaction of the intermediate with solvent water to that for a second reaction which serves as a clock. The rate constants for these clock reactions are known with good precision, because they are limited by the rate constant for a physical transport step which is largely independent of the structure of the intermediate.
Introduction
The generation of most localized and many delocalized carbanions and carbocations in water is problematic, because the chemical barrier to their Brnsted base or Lewis acid reactions with solvent is often smaller than that for a bond vibration.1 An important challenge to laboratory chemists and living systems is the development of methodology for cleavage and synthesis of carbon-carbon bonds through carbanion or carbocation intermediates. The formation of these species is often facilitated by delocalization of charge away from carbon and onto distant electronegative atoms. However, these ions are referred to as carbocations and carbanions even when the bulk of the charge lies distant from the reacting carbon, because the preponderance of their chemical reactions occur at carbon. Such resonance-stabilized carbocations and carbanions are ubiquitous intermediates of metabolic and synthetic organic reactions, and the study of their
Scheme 1
John P. Richard received a B.S. degree in biochemistry and his Ph.D. degree in chemistry (1979) from The Ohio State University. He is currently Professor of Chemistry at the University at Buffalo, SUNY. Tina L. Amyes received B.A. and M.A. degrees in natural sciences and her Ph.D. degree in chemistry (1986) from the University of Cambridge (England). She is currently an Adjunct Assistant Professor at the University at Buffalo, SUNY. Maria M. Toteva received B.S. and M.S. degrees in chemistry from Sofia University (Bulgaria) and her Ph.D. degree in chemistry (1999) from the University at Buffalo, SUNY. She is currently a Senior Scientist at the University at Buffalo, SUNY.
10.1021/ar0000556 CCC: $20.00 Published on Web 10/24/2001 2001 American Chemical Society
Clocks for Carbocations. There are sharp decreases in the rate constant ratio kaz/ks (M-1) for partitioning of 1-arylethyl and 1-aryl-2,2,2-trifluoroethyl carbocations between nucleophilic addition of azide ion, kaz (M-1 s-1, Scheme 1), and solvent, ks (s-1), when the carbocation is destabilized by the addition of electron-withdrawing aromatic ring substituents.3,4 This is because kaz is the invariant rate constant for a diffusion-limited reaction, but ks is limited by the chemical barrier to solvent addition and increases with decreasing carbocation stability. Values of ks for reaction of a variety of carbocations with aqueous solvents have been obtained from kaz/ks (M-1) determined by product analysis (eq 1), using kaz ) 5 109 M-1 s-1 (Scheme 1).3,4 In cases where a comparison is possible, provided kaz/ks e 104 M-1, the values of ks from this azide
* Phone: (716) 645-6800 ext 2194. Fax: (716) 645-6963. E-mail: jrichard@chem.buffalo.edu.
VOL. 34, NO. 12, 2001 / ACCOUNTS OF CHEMICAL RESEARCH 981
Carbocations and Carbanions in Water Richard et al.
ion clock are in excellent agreement ((20%) with those determined directly by generation of the same carbocation by laser flash photolysis.2a,5
kaz/ks )
[RN3] [ROH][N3-]
(1) (2) (3)
kaz ) 5 109 M-1 s-1 pKR ) -log KR ) log(kH/ks)
Absolute rate constants for carbocation-nucleophile addition have been used to calculate equilibrium constants for carbocation formation. (1) Values of ks (s-1) have been combined with rate constants kH (M-1 s-1) for acidcatalyzed cleavage of the corresponding alcohols to give values of pKR for R-substituted benzyl, benzhydryl, and 9-fluorenyl carbocations (eq 3, Scheme 1).6 (2) Rate constants ksolv (s-1) for the stepwise solvolysis of gemdiazides, R-alkoxy azides, and R-substituted benzylic azides through carbocation intermediates have been determined and combined with kaz ) 5 109 M-1 s-1 for the diffusion-controlled capture of these carbocations by azide ion to give equilibrium constants Kaz ) ksolv/kaz (M) for formation of the carbocation from the neutral azide ion adduct (Scheme 1).7 Clocks for Ion Pairs. The estimated value of k-d ) 1.6 1010 s-1 for diffusional separation of carbocation-anion ion pairs to give the free ions in 50:50 (v/v) water/ trifluoroethanol8 serves as a useful clock for other reactions of ion pairs. We have used this clock to determine a number of rate constants that describe the dynamics of these short-lived intermediates. (1) Reaction of 1-(4-thiomethylphenyl)ethyl thionobenzoate (1, X ) S, Y ) O, Z ) MeS, Ar ) C6H5) in 50:50 (v/v) water/trifluoroethanol gives 84% of the rearranged thiolbenzoate ester 2 and 14% of the solvent adducts 3 (Scheme 2).9 It was shown that kc > k-r and k-d . ks, so that the product ratio [2]/[3] ) 84/16 corresponds to kr/ k-d ) 6 for partitioning of the ion pair intermediate between reorganization that exchanges the positions of O and S at the leaving group anion and diffusional separation to give the free ions.9 This was combined with
k-d ) 1.6 1010 s-1 to give kr ) 1011 s-1 as the absolute rate constant for reorganization of the ion pair intermediate within an aqueous solvation shell (Scheme 2).9 (2) The rate constant ratios for partitioning of the ion pair intermediate generated by cleavage of 4-MeC6H413 CH(Me)18OC(16O)C6F5 (1, X ) 16O, Y ) 18O, Z ) Me, Ar ) C6F5) between diffusional separation (k-d) and direct nucleophilic addition of solvent (ks) to give 3, and internal return with 18O scrambling (kc) to give the isomerization product 2, have been determined and combined with the known values of k-d and ks to give kc ) 7 109 s-1 (kc < kr, Scheme 2) for unimolecular collapse of the ion pair intermediate (Scheme 2).10 This relatively large value of kc shows that kNu ) 5 108 M-1 s-1 for bimolecular nucleophilic addition of carboxylate anions to the free carbocation 4-MeC6H4CH(Me)+ is limited by the partial desolvation of the carboxylate anion that is required for formation of an intimate ion pair.8 Clocks for Carbanions. There are several cases for which proton transfer to a carbanion is limited by the physical transport of the reacting acid into a reactive position with a known rate constant. These physical processes serve as clocks and have been used to determine the lifetimes of simple carbanions and to obtain reliable carbon acid pKas for their formation in water.
Scheme 2
The limiting rate constant (kHOH)lim (s-1) for protonation of carbanions by solvent water is that for rotation of a molecule of water into a position where proton transfer to the carbanion can occur. It is given by the rate constant for the dielectric relaxation (reorganization) of solvent water, (kHOH)lim ) kreorg 1011 s-1.11 Therefore, solvent reorganization serves as a clock for the reactions of carbanions which are formed in water but which exist in this solvent only for the time required for solvent reorganization. The primary deuterium isotope effect on the hydroxide-ion-catalyzed exchange of hydrons from water into acetonitrile and succinonitrile that proceeds through the R-cyano carbanions 4a and 4b, respectively, is not much larger than unity.11 This provides good evidence that the isotope-dependent chemical barrier to protonation of these carbanions by solvent water (kHOL) is smaller than the isotope-independent barrier to solvent reorganization (kreorg), so that solvent reorganization is rate-limiting for kHOL (Scheme 3).11 A carbon acid pKa of 28.9 for acetonitrile in water was calculated from the experimental rate constants kHO (M-1 s-1) for deprotonation of the carbon acid by hydroxide ion and kHOH ) (kHOH)lim ) 1011 s-1 for carbanion protonation (eq 4 derived for Scheme 3).11
Scheme 3
982 ACCOUNTS OF CHEMICAL RESEARCH / VOL. 34, NO. 12, 2001
pKa ) pKw + log
( )
kHOH kHO
(4)
Scheme 4
Figure 1 shows the linear correlations of log kHOH (s-1) for protonation and log kHO (M-1 s-1) for deprotonation, with slopes of +0.60 and -0.40, respectively, with the pKas of neutral R-carbonyl carbon acids in water (Scheme 3).12-14 The values of kHOH increase with decreasing carbanion stability to kHOH 109 s-1 for protonation of the enolate of acetamide (5),14 but must level off at kHOH ) (kHOH)lim 1011 s-1 when proton transfer is limited by solvent reorganization. A short extrapolation of the linear
correlation for log kHOH shows that this leveling will occur for an enolate that is formed by deprotonation of a carbon acid of pKa 30. We have determined kDO ) 8.4 10-9 M-1 s-1 for deuterioxide-ion-catalyzed exchange for deuterium from D2O of the R-protons of acetate ion.14 If isotope exchange proceeds through the enolate dianion, then we estimate kHO ) 3.5 10-9 M-1 s-1 for deprotonation of acetate ion by hydroxide ion (Scheme 3).14 However, this value of kHO cannot lie on the line obtained by linear extrapolation of the correlation for log kHO (Figure 1, O), because this would require pKa ) 38.0 for acetate ion and kHOH ) 3 1015 s-1 (Figure 1, 0) for protonation of the enolate dianion by solvent water! The extrapolated lifetime of the putative enolate dianion intermediate is even shorter than the time for a bond vibration (10-13 s).1 Therefore, if the enolate dianion forms as an intermediate of the deuterium exchange reaction of acetate ion, then kHOH ) (kHOH)lim ) 1011 s-1 for its protonation by solvent water, which gives pKa ) 33.5 for acetate ion (eq 4).14 The Brnsted coefficient ) 1.09 was determined for exchange for deuterium from D2O of the R-protons of
ethyl acetate catalyzed by 3-substituted quinuclidines, which proceeds through the ester enolate 6.13 This shows that there is complete proton transfer from the carbon acid to the amine base in the transition state for deuterium exchange, and it requires that the rate-determining step for formation of the free enolate 6 (kB, Scheme 4) be the diffusional separation of a reversibly formed enolateammonium ion complex. Therefore, the reverse protonation of 6 by ammonium cations is limited by the essentially diffusion-controlled encounter of these ions, with kBH 5 109 M-1 s-1.13 The enolate of acetamide, 5, is even less stable than 6 and also undergoes diffusionlimited protonation by buffer acids.14 The second-order rate constants kB for deprotonation of ethyl acetate and acetamide by quinuclidine (pKBH ) 11.5) were combined with kBH ) kd 5 109 M-1 s-1 for diffusion-limited protonation of the enolate (eq 5 derived for Scheme 4) to give pKas of 25.6 and 28.4, respectively, for these carbon acids in water.13,14
pKa ) pKBH + log
( )
kBH kB
(5)
The limiting rate constant for protonation of Brnsted bases by hydronium ion is kH 1010 M-1 s-1,15 which is close to kH ) 7 109 M-1 s-1, reported for protonation of the enolate of acetone.16 There is evidence that, in water, the zwitterionic enolate of betaine methyl ester is only slightly more stable than the enolate of acetone, so that kH ) 5 109 M-1 s-1 was estimated for its protonation by hydronium ion (Scheme 5).12 This was combined with the experimental rate constant kw ) 5 10-9 s-1 for deprotonation of betaine methyl ester by solvent water to give a carbon acid pKa of 18.0 for betaine methyl ester (pKa ) log{kH/kw}, Scheme 5).12
Scheme 5
FIGURE 1. Rate-equilibrium correlations for deprotonation of neutral R-carbonyl carbon acids by hydroxide ion, kHO (b, M-1 s-1), and the reverse protonation of the enolates by solvent water, kHOH (9, s-1), with the pKa of the carbon acid. (O, 0) Extrapolated data for proton-transfer reactions of acetate ion and its enolate dianion (see text).
Reliable estimates of the carbon acid pKas in water for ethyl thiolacetate (pKa ) 21.0),17 N-protonated glycine methyl ester (+H3NCH2CO2Me, pKa ) 21.0),12,18 glycine zwitterion (+H3NCH2CO2-, pKa ) 28.9),12 and betaine (+Me3NCH2CO2-, pKa ) 27.3)12 were obtained by comparison of experimentally determined rate constants for deprotonation of these carbon acids with those for deprotonation of structurally related carbon acids of known pKa. In general, reliable pKas of simple neutral R-carbonyl carbon acids may be obtained from kHO (M-1 s-1) for deprotonation of the carbon acid by interpolation of the excellent linear correlation between log kHO and pKa shown in Figure 1.13,14
FIGURE 2. Free energy reaction coordinate profiles for protonation of the steroid enolate 7- emphasizing the difference in the products formed under conditions of kinetic and thermodynamic control.
FIGURE 3. Free energy reaction coordinate profiles for addition of nucleophiles to carbocations. (A) Essentially barrierless addition of nucleophiles to the methyl carbocation. (B) Thermoneutral addition of nucleophiles to a resonance-stabilized carbocation with an intrinsic barrier .
electron-donating substituents will result in a change to a thermoneutral reaction when the stabilization resulting from formation of the new bond to the nucleophile is exactly balanced by the destabilization resulting from the loss of resonance electron donation to the cationic center (Figure 3B). An intrinsic barrier for this thermoneutral nucleophile addition to a resonance-stabilized carbocation will be observed when the energetic cost of the loss of the stabilizing resonance interactions at the transition state exceeds the compensating transition state stabilization resulting from partial carbon-nucleophile bond formation,22 so that there is an imbalance in the changes in these opposing interactions. The work of C. F. Bernasconi has provided many examples of correlations between the increase in the intrinsic barrier to protonation of carbanions with increasing stabilization of the carbanion by resonance delocalization of negative charge and the requirement for a relatively large or early loss of these stabilizing resonance interactions in the transition state.23 This rapid falloff in resonance stabilization with formation of a partial covalent bond to sp2-hybridized carbon is proposed to result from (1) a decrease in the stabilizing resonance interaction with increasing bond formation, which is expected to be roughly proportional to the fractional formation of the new covalent bond, and (2) an additional falloff in the remaining resonance interaction, which is due to the change from sp2 hybridization of the planar central carbon, for which there is optimal delocalization of charge, toward sp3 hybridization at the transition state.3,24,25 The defining property of most carbocations and carbanions with significant lifetimes in water is the large stabilization of these ions by delocalization of charge away from the central carbon and onto distant atoms. However, a number of other interactions are known to affect the thermodynamic driving force associated with formation of chemical bonds to carbocations and carbanions, including geminal interactions between electron-withdrawing substituents at sp3-hybridized carbon,26 steric interactions,27 and differential solvation of reactants and
Kinetic and Thermodynamic Substituent Effects

The products of competing reactions of organic ions formed under conditions of kinetic control are sometimes different from those formed under conditions of thermodynamic control. Figure 2 illustrates this for protonation of the delocalized steroid enolate ion 7-, which gives mainly 7-(r-H) under conditions of kinetic control and mainly 7-(-H) under conditions of thermodynamic control.19 This difference in the products of reactions under kinetic and thermodynamic control shows that protonation of 7- at C-R is intrinsically easier than its protonation at C- and, therefore, occurs with a smaller Marcus intrinsic barrier for thermoneutral proton transfer (, Scheme 6).20 The reaction is under kinetic control, because the effect of the smaller intrinsic barrier for protonation of 7- at C-R overwhelms the greater thermodynamic driving force G for its protonation at C-. This notion that a substituent effect on may be different from, and dominate over, the substituent effect on the thermodynamic driving force was suggested over 30 years ago by R. A. Marcus in his explanation for the unusually large Brnsted coefficient for deprotonation of aryl substituted nitroalkanes by carboxylate anions.21 It is a recurring theme from our own work, and that of others, that is deserving of wider recognition within the community of organic chemists.
Scheme 6
Origin of and Changes in Intrinsic Barriers

Consider the hypothetical addition of a nucleophile to the methyl carbocation, a reaction which is strongly favored thermodynamically and has essentially no activation barrier (Figure 3A). The stabilization of CH3+ by resonance
products.23 Any such interaction which stabilizes product relative to reactant will result in an increase in the intrinsic barrier when the fractional expression of the interaction at the transition state is small and a decrease in when its expression is large, relative to bond making/breaking at the reacting central carbon.23
Resonance Effects
Much of the difficulty experienced in interpreting structure-reactivity effects on the reaction of resonancestabilized carbocations28 and carbanions arises from the failure to appreciate fully the complexity of resonance effects on these reactions. The results of our work suggest two generalizations about these resonance substituent effects. (1) The stabilization of carbocations or carbanions by resonance delocalization of positive or negative charge is not constant but, rather, is attenuated by polar substituents attached to the charged center. For example, the addition of polar electron-withdrawing R-substituents to the 4-methoxybenzyl carbocation (Scheme 7A) enhances resonance electron donation from the aromatic ring. This increases the separation between the interacting positive charge and dipole(s) and therefore reduces their destabilizing interaction. By contrast, the addition of polar electron-withdrawing substituents to the R-carbon of an enolate reduces the resonance delocalization of negative charge onto the carbonyl group. This results in an increase in the localization of negative charge at the R-carbon, thereby increasing its stabilizing interaction with the polar substituent (Scheme 7B). (2) The fractional loss of resonance stabilization from electron-donating R-substituents in the transition state for the reactions of carbocations may not always be constant but may vary with the nature of the electron-donating substituent. These two generalizations are useful in explaining several of the following results.
from attenuation of destabilizing polar interactions at the cationic center by the enhancement of resonance delocalization of the positive charge onto the 4-methoxyphenyl ring (Scheme 7A).32 This results in an increase in the intrinsic barrier for nucleophile addition and a decrease in ks which opposes the increase in ks that is due to the polar substituent effect on the thermodynamic driving force.4,7,28 An R-OR carbocation is generally less stable thermodynamically than the corresponding R-SR carbocation, but it is both formed more rapidly in solvolysis reactions and reacts more rapidly with nucleophilic solvent.33 This shows that there is a smaller intrinsic barrier to the formation and reaction of R-OR compared with R-SR stabilized carbocations. We have proposed that this is the result of greater stabilization of the partially sp3-hybridized cationic center in the transition state for solvent addition by electron donation from an R-OR substituent (8, X ) O) than from an R-SR substituent (8, X ) S)33 and have provided solid theoretical justification for this proposal.28
Scheme 7
Carbocations. The rate constants ks for addition of a largely aqueous solvent to R-substituted 4-methoxybenzyl carbocations remain nearly constant as the thermodynamic driving force for nucleophile addition is varied by 23 kcal/mol (!) by the addition of polar electron-withdrawing R-substituents (Scheme 7A).4,7,29-31 We have proposed that these nearly invariant rate constants result
A comparison of the effect of R-oxygen, R-azido, and R-(4-methoxyphenyl) substituents on the equilibrium constant Kaz ) ksolv/kaz for formation of carbocations from the neutral azide ion adduct (Scheme 1) and on ks for nucleophilic addition of solvent water shows that the R-oxygen substituent provides the largest thermodynamic stabilization of a simple primary carbocation, but that it also results in the smallest kinetic barrier associated with loss of the stabilizing resonance interactions in the transition state for nucleophile addition (Table 1).34 In other words, nucleophilic addition to R-oxygen carbocations is easier and proceeds over a smaller intrinsic barrier than addition to R-azido and R-(4-methoxyphenyl) carbocations. The order of reactivity of these carbocations (Table 1) shows that resonance electron donation to a partly filled -orbital at the cationic center in the transition state for nucleophile addition (see 8) is maximal for direct electron donation from an R-ethoxy group and decreases as the number of atoms through which the effect is expressed is increased on moving to the R-azido and R-(4methoxyphenyl) groups.34 The geminal interaction between first-row electronegative atoms at sp3-hybridized carbon (9) is stabilizing by up to 17 kcal/mol.35 We have shown that substrates with a strong stabilizing geminal interaction at the central reacting carbon, such as methoxymethyl fluoride (MeOCH2F), are strongly stabilized toward solvolysis in comparison with substrates such as 4-methoxybenzyl fluoride.26 A large fractional development of stabilizing geminal interactions in the transition state for addition of oxygen nucleophiles to R-oxygen stabilized carbocations might contribute to the smaller intrinsic barrier
Table 1. Effects of Strongly Electron-Donating r-Substituents on the Thermodynamic Stability of Primary Alkyl Carbocations and on the Rate Constant for Nucleophilic Addition of Solvent Water (Scheme 1)a
a In water at 25 C. b G az ) -RT ln Kaz (Scheme 1). Values of Kaz ) ksolv/kaz were taken from ref 34. c Rate constant for nucleophilic addition of solvent water taken from ref 34.
for nucleophile addition to R-OR stabilized carbocations compared with R-SR and R-(4-methoxyphenyl) stabilized carbocations.33,34 However, the expression of these geminal interactions at the transition state for nucleophilic addition to carbocations has not been evaluated.
occurs with increasing pKa of these R-carbonyl carbon acids is due to a decrease in the resonance stabilization of the enolate product, which is also expected to lead to a decrease in the Marcus intrinsic barrier for proton transfer.23 We have therefore proposed that the change to a steeper, more negative, slope of the correlation in Figure 1 with increasing thermodynamic barrier to carbon deprotonation that is predicted by the Marcus equation is masked by opposing increases in kHO resulting from concomitant decreases in .13 The intrinsic barrier for protonation of R-cyano carbanions is substantially smaller than that for protonation of enolates and other resonance-stabilized carbanions,36 but there has been little consideration of the broader implication of this small barrier. We have found it useful to compare C-protonation of the cyanomethyl carbanion (Scheme 8) with O-protonation of acetate ion.15 The small intrinsic barrier to O-protonation of acetate ion reflects the small stabilization associated with delocalization of charge between the two oxygens.37 There is a similar small intrinsic barrier associated with C-protonation of R-cyano carbanions (kHOH, Scheme 8),11 where the charge is distributed approximately equally between carbon (60%) and the R-cyano group (40%)38 and the stabilization associated with charge delocalization is presumably small.
Scheme 8
Absolute and Relative Magnitudes of Intrinsic Barriers to Organic Reactions

Carbanions. The addition of cationic R-NH3+ or R-NMe3+ substituents to the enolate of ethyl acetate results in a decrease in the intrinsic barrier for protonation of the carbanion by solvent water (Scheme 7B).12 We have proposed that there is a relationship between this decrease in and the tendency of these charged R-substituents to lead to an increase in the localization of the negative charge at the R-amino carbon, which enhances the stabilizing polar interaction between unlike charges at the zwitterionic enolate (Scheme 7B).12 Figure 1 (b) shows the good linear correlation (slope ) -0.40) up to pKa 30 between log kHO for (mostly) thermodynamically unfavorable deprotonation by hydroxide ion and the pKa of neutral R-carbonyl carbon acids (Scheme 3).13,14 This extended linear relationship is surprising, because proton-transfer reactions are expected to show a change to a more productlike transition state with decreasing thermodynamic driving force (increasing carbon acid pKa), which should result in downward curvature and an absolute slope of >|-0.5| for unfavorable proton transfer. This curvature is predicted by different forms of the Marcus equation, but the data in Figure 1 do not show satisfactory fits to these models.13 Much of the change in the thermodynamic driving force for proton transfer that
We have determined absolute and relative intrinsic barriers for several organic reactions and have offered sensible interpretations of these data. These determinations are limited; our interpretations are complex and undergoing constant evaluation. Input from the organic community would be valued. Nucleophile Addition to a Quinone Methide. The reactivity of the quinone methide 10 toward nucleophiles is similar to that of other strongly resonance-stabilized carbocations.22,39 There is no simple relationship between the rate and equilibrium constants Keq ) kNu/ksolv (M-1) for addition of halide and acetate ions to 10 (Scheme 9), because there are differences of up to 7 kcal/mol in the Marcus intrinsic barrier for addition of different nucleophiles: Nu-, (kcal/mol) I-, 12.4; Br-, 13.9; Cl-, 15.4; AcO-, 19.8.22 Similar differences in are observed for addition of halide ions to the trityl carbocation.22
Scheme 9
The R-deuterium isotope effect for concerted bimolecular nucleophilic substitution at N-(methoxymethyl)-
N,N-dimethylanilinium ions decreases with increasing hardness of the nucleophile: Nu-, kH/kD I-, 1.18; Br-, 1.16; Cl-, 1.13; AcO-, 1.07.40 Therefore, the hybridization of the central carbon in the transition state for nucleophilic substitution by iodide ion is close to that for the methoxymethyl carbocation (MeOCH2+), and there is a shift to a transition state with greater bonding between the nucleophile and the central carbon on moving along the series I-, Br-, Cl-, AcO-. These results suggest that the transition state for nucleophile addition to 10 and the trityl carbocation is reached earlier (more cationic), and with a lower intrinsic barrier, for soft strongly polarizable nucleophiles such as iodide ion compared with hard weakly polarizable nucleophiles such as acetate ion, because the former nucleophiles provide greater orbital overlap at longer bond distances.22 If different degrees of bond formation are observed at the transition state for thermoneutral addition of different nucleophiles to 10, then the intersecting curves that constitute the reaction coordinate profile for nucleophile addition must in some cases exhibit pronounced asymmetry (Scheme 10).
Scheme 11
Scheme 12
Scheme 10
We have examined the addition of thiols and sulfides to 10.27 The polarizable methyl group at dimethyl sulfide provides significant stabilization of the charge at the cationic sulfur of the nucleophile addition product, but it creates an even larger destabilizing steric/electrostatic interaction with the R-CF3 groups. The reaction proceeds through a transition state at which there is an imbalance between the large expression of the stabilizing interactions of the polarizable methyl group and the smaller expression of destabilizing steric/electrostatic interactions.27 These data suggest that bonding interactions between 10 and Me2S in the transition state for nucleophile addition are expressed at a large distance, which minimizes steric/ electrostatic interactions between the nucleophile and the R-CF3 groups, so that these destabilizing interactions develop after the transition state has been traversed on the reaction coordinate. Reactions of Oxocarbenium Ions. The intrinsic barrier for nucleophilic addition of methanol to the oxocarbenium ion 11H+ to give 12H+, MeOH ) 6.5 kcal/mol, is about 50% smaller than that for deprotonation of 11H+ at carbon by solvent water to give 11, p ) 13.8 kcal/mol (Scheme 11).41 The difference in these intrinsic barriers may be related to the requirement for more extensive electronic reorganization on proceeding to the transition state for deprotonation of 11H+ compared with the transition state for direct nucleophile addition to the carbocation.41
Aldol Addition Reactions. The requirement for rehybridization and electronic reorganization at the benzaldehyde carbonyl group upon formation of the tetrahedral adduct with hydroxide ion results in an 8 kcal/mol larger Marcus intrinsic barrier compared with that for direct proton transfer between hydroxide ion and water.42 Similarly then, the intrinsic barrier for addition of an enolate ion to the benzaldehyde carbonyl group (kc, Scheme 12) might also be expected to be larger than that for protonation of the enolate by solvent water (kHOH, Scheme 12). In fact, we have shown that these processes have very similar Marcus intrinsic barriers of ) 14.1 kcal/mol for kc and ) 14.7 kcal/mol for kHOH.42 This suggests that there may be compensating stabilization of the transition state for aldol addition by favorable interactions between the HOMO of the soft enolate nucleophile and the LUMO of the soft carbonyl group electrophile.42
This work is the result of the training that J.P.R. and T.L.A. received as Postdoctoral Fellows in the laboratory of William P. Jencks at Brandeis University and the efforts of their co-workers at Kentucky and Buffalo. Our work has been generously supported by Grant GM39754 from the National Institutes of Health.
References
(1) Jencks, W. P. How Does a Reaction Choose its Mechanism? Chem. Soc. Rev. 1981, 10, 345-375. (2) (a) McClelland, R. A. Flash Photolysis Generation and Reactivities of Carbenium Ions and Nitrenium Ions. Tetrahedron 1996, 52, 6823-6858. (b) Chiang, Y.; Kresge, A. J. Enols and Other Reactive Species. Science 1991, 253, 395-400. (3) Richard, J. P.; Rothenberg, M. E.; Jencks, W. P. Formation and Stability of Ring-Substituted 1-Phenylethyl Carbocations. J. Am. Chem. Soc. 1984, 106, 1361-1372. (4) Richard, J. P. The Extraordinarily Long Lifetimes and Other Properties of Highly Destabilized Ring-Substituted 1-Phenyl-2,2,2trifluoroethyl Carbocations. J. Am. Chem. Soc. 1989, 111, 14551465. (5) McClelland, R. A.; Cozens, F. L.; Steenken, S.; Amyes, T. L.; Richard, J. P. Direct Observation of -Fluoro-substituted 4-Methoxyphenethyl Cations by Laser Flash Photolysis. J. Chem. Soc., Perkin Trans. 2 1993, 1717-1722.

(6) Amyes, T. L.; Richard, J. P.; Novak, M. Experiments and Calculations for Determination of the Stabilities of Benzyl, Benzhydryl, and Fluorenyl Carbocations: Antiaromaticity Revisited. J. Am. Chem. Soc. 1992, 114, 8032-8041. (7) Amyes, T. L.; Stevens, I. W.; Richard, J. P. The Effects of R-Substituents on the Kinetic and Thermodynamic Stability of 4-Methoxybenzyl Carbocations: Carbocation Lifetimes That Are Independent of Their Thermodynamic Stability. J. Org. Chem. 1993, 58, 6057-6066. (8) Richard, J. P.; Jencks, W. P. Reactions of Substituted 1-Phenylethyl Carbocations with Alcohols and Other Nucleophilic Reagents. J. Am. Chem. Soc. 1984, 106, 1373-1383. (9) Richard, J. P.; Tsuji, Y. Dynamics for Reaction of an Ion Pair in Aqueous Solution: The Rate Constant for Ion Pair Reorganization. J. Am. Chem. Soc. 2000, 122, 3963-3964. (10) Tsuji, Y.; Mori, T.; Richard, J. P.; Amyes, T. L.; Fujio, M.; Tsuno, Y. Dynamics for Reaction of an Ion Pair in Aqueous Solution: Reactivity of Carboxylate Anions in Bimolecular CarbocationNucleophile Addition and Unimolecular Ion Pair Collapse. Org. Lett. 2001, 3, 1237-1240. (11) Richard, J. P.; Williams, G.; Gao, J. Experimental and Computational Determination of the Effect of the Cyano Group on Carbon Acidity in Water. J. Am. Chem. Soc. 1999, 121, 715-726. (12) Rios, A.; Amyes, T. L.; Richard, J. P. Formation and Stability of Organic Zwitterions in Aqueous Solution: Enolates of the Amino Glycine and Its Derivatives. J. Am. Chem. Soc. 2000, 122, 93739385. (13) Amyes, T. L.; Richard, J. P. Determination of the pKa of Ethyl Acetate: Brnsted Correlation for Deprotonation of a Simple Oxygen Ester in Aqueous Solution. J. Am. Chem. Soc. 1996, 118, 3129-3141. (14) Richard, J. P.; Williams, G.; ODonoghue, A. C.; Amyes, T. L. Formation and Stability of Enolates of Acetamide and Acetate Ion: An Eigen Plot for Proton Transfer at R-Carbonyl Carbon. J. Am. Chem. Soc., submitted for publication. (15) Eigen, M. Proton Transfer, Acid-Base Catalysis, and Enzymatic Hydrolysis. Angew. Chem., Int. Ed. Engl. 1964, 3, 1-72. (16) Keeffe, J. R.; Kresge, A. J. In The Chemistry of Enols; Rappoport, Z., Ed.; John Wiley and Sons: Chichester, 1990; pp 399-480. (17) Amyes, T. L.; Richard, J. P. Generation and Stability of a Simple Thiol Ester Enolate in Aqueous Solution. J. Am. Chem. Soc. 1992, 114, 10297-10302. (18) Rios, A.; Richard, J. P. Biological Enolates: Generation and Stability of the Enolate of N-Protonated Glycine Methyl Ester in Water. J. Am. Chem. Soc. 1997, 119, 8375-8376. (19) Malhotra, S. K.; Ringold, H. J. Chemistry of Conjugate Anions and Enols. IV. The Kinetically Controlled Enolization of R,-Unsaturated Ketones and the Nature of the Transition State. J. Am. Chem. Soc. 1964, 86, 1997-2003. (20) Marcus, R. A. Theoretical Relations among Rate Constants, Barrier, and Brnsted Slopes of Chemical Reactions. J. Phys. Chem. 1968, 72, 891-899. (21) Marcus, R. A. Unusual Slopes of Free Energy Plots in Kinetics. J. Am. Chem. Soc. 1969, 91, 7224-7225. (22) Richard, J. P.; Toteva, M. M.; Crugeiras, J. Structure-Reactivity Relationships and Intrinsic Reaction Barriers for Nucleophile Additions to a Quinone Methide: A Strongly ResonanceStabilized Carbocation. J. Am. Chem. Soc. 2000, 122, 1664-1674. (23) Bernasconi, C. F. The Principle of Nonperfect Synchronization: More Than a Qualitative Concept? Acc. Chem. Res. 1992, 25, 9-16. (24) Kresge, A. J. The Nitroalkane Anomaly. Can. J. Chem. 1974, 52, 1897-1903. (25) Amyes, T. L.; Jencks, W. P. Lifetimes of Oxocarbenium Ions in Aqueous Solution from Common Ion Inhibition of the Solvolysis of R-Azido Ethers by Added Azide Ion. J. Am. Chem. Soc. 1989, 111, 7888-7900. (26) Richard, J. P.; Amyes, T. L.; Rice, D. J. Effects of Electronic Geminal Interactions on the Solvolytic Reactivity of Methoxymethyl Derivatives. J. Am. Chem. Soc. 1993, 115, 2523-2524. (27) Toteva, M. M.; Richard, J. P. Structure-Reactivity Relationships for Addition of Sulfur Nucleophiles to Electrophilic Carbon: Resonance, Polarization, and Steric/Electrostatic Effects. J. Am. Chem. Soc. 2000, 122, 11073-11083. (28) Richard, J. P. A Consideration of the Barrier for CarbocationNucleophile Combination Reactions. Tetrahedron 1995, 51, 15351573. (29) Richard, J. P.; Amyes, T. L.; Bei, L.; Stubblefield, V. Effect of -Fluorine Substituents on the Rate and Equilibrium Constants for the Reactions of R-Substituted 4-Methoxybenzyl Carbocations and on the Reactivity of a Simple Quinone Methide. J. Am. Chem. Soc. 1990, 112, 9513-9519. (30) Richard, J. P.; Amyes, T. L.; Stevens, I. W. Generation and Determination of the Lifetime of an R-Carbonyl Substituted Carbocation. Tetrahedron Lett. 1991, 32, 4255-4258. (31) Richard, J. P.; Lin, S.-S.; Buccigross, J. M.; Amyes, T. L. How Does Organic Structure Determine Organic Reactivity? Nucleophilic Substitution and Alkene-Forming Elimination Reactions of R-Carbonyl and R-Thiocarbonyl Substituted Benzyl Derivatives. J. Am. Chem. Soc. 1996, 118, 12603-12613. (32) Lee, I.; Chung D. S.; Jung, J. H. Theoretical Studies of the Effects of R-Substituents on the Resonance Demand of 4-Methoxybenzyl Carbocations. Tetrahedron 1994, 50, 7981-7986. (33) Jagannadham, V.; Amyes, T. L.; Richard, J. P. Kinetic and Thermodynamic Stabilities of R-Oxygen- and R-Sulfur-Stabilized Carbocations in Solution. J. Am. Chem. Soc. 1993, 115, 84658466. (34) Richard, J. P.; Amyes, T. L.; Jagannadham, V.; Lee, Y.-G.; Rice, D. J. Spontaneous Cleavage of gem-Diazides: A Comparison of the Effects of R-Azido and Other Electron-Donating Groups on the Kinetic and Thermodynamic Stability of Benzyl and Alkyl Carbocations in Aqueous Solution. J. Am. Chem. Soc. 1995, 117, 5198-5205. (35) Schleyer, P. v. R. A Systematic Survey of the Effects of Substituents on the Structures and Energies of the Principal Organic Reaction Intermediates. Pure Appl. Chem. 1987, 59, 1647-1660. (36) Hojatti, M.; Kresge, A. J.; Wang, W.-H. Cyanocarbon Acids: Direct Evidence That Their Ionization Is Not an Encounter-Controlled Process and Rationalization of the Unusual Solvent Isotope Effects. J. Am. Chem. Soc. 1987, 109, 4023-4028. (37) Rablen, P. R. Is the Acetate Anion Stabilized by Resonance or Electrostatics? A Systematic Structural Comparison. J. Am. Chem. Soc. 2000, 122, 357-368. (38) Wiberg, K. B.; Castejon, H. Carbanions 2. Intramolecular Interactions in Carbanions Stabilized by Carbonyl, Cyano, Isocyano, and Nitro Groups. J. Org. Chem. 1995, 60, 6327-6334. (39) Richard, J. P. Mechanisms for the Uncatalyzed and Hydrogen Ion Catalyzed Reactions of a Simple Quinone Methide with Solvent and Halide Ions. J. Am. Chem. Soc. 1991, 113, 4588-4595. (40) Knier, B. L.; Jencks, W. P. Mechanism of Reactions of N-(Methoxymethyl)-N, N-Dimethylanilinium Ions with Nucleophilic Reagents. J. Am. Chem. Soc. 1980, 102, 6789-6798. (41) Richard, J. P.; Williams, K. B.; Amyes, T. L. Intrinsic Barriers for the Reactions of an Oxocarbenium Ion in Water. J. Am. Chem. Soc. 1999, 121, 8403-8404. (42) Richard, J. P.; Nagorski, R. W. Mechanistic Imperatives for Catalysis of Aldol Addition Reactions: Partitioning of the Enolate Intermediate between Reaction with Brnsted Acids and the Carbonyl Group. J. Am. Chem. Soc. 1999, 121, 4763-4770.
AR0000556
Phosphate Binding Energy and Catalysis by Small and Large Molecules

JANET R. MORROW, TINA L. AMYES, AND JOHN P. RICHARD*
Department of Chemistry, University at Buffalo, State University of New York, Buffalo, New York 14260-3000
RECEIVED ON SEPTEMBER 11, 2007
CON SPECTUS
atalysis is an important process in chemistry and enzymology. The rate acceleration for any catalyzed reaction is the difference between the activation barriers for the uncatalyzed (GHOq) and catalyzed (GMeq) reactions, which corresponds to the binding energy (GSq ) GMeq - GHOq) for transfer of the reaction transition state from solution to the catalyst. This transition state binding energy is a fundamental descriptor of catalyzed reactions, and its evaluation is necessary for an understanding of any and all catalytic processes. We have evaluated the transition state binding energies obtained from interactions between low molecular weight metal ion complexes or high molecular weight protein catalysts and the phosphate group of bound substrate. Work on catalysis by small molecules is exemplied by studies on the mechanism of action of Zn2(1)(H2O). A binding energy of GSq ) -9.6 kcal/mol was determined for Zn2(1)(H2O)-catalyzed cleavage of the RNA analogue HpPNP. The pH-rate prole for this cleavage reaction showed that there is optimal catalytic activity at high pH, where the catalyst is in the basic form [Zn2(1)(HO-)]. However, it was also shown that the active form of the catalyst is Zn2(1)(H2O) and that this recognizes the C2-oxygenionized substrate in the cleavage reaction. The active catalyst Zn2(1)(H2O) shows a high afnity for oxyphosphorane transition state dianions and a stable methyl phosphate transition state analogue, compared with the afnity for phosphate monoanion substrates. The transition state binding energies, GSq, for cleavage of HpPNP catalyzed by a variety of Zn2+ and Eu3+ metal ion complexes reect the increase in the catalytic activity with increasing total positive charge at the catalyst. These values of GSq are affected by interactions between the metal ion and its ligands, but these effects are small in comparison with GSq observed for catalysis by free metal ions, where the ligands are water. Enzymes are unique in having evolved mechanisms to effectively utilize binding interactions with nonreacting fragments of the substrate in stabilization of the reaction transition state. Orotidine 5-monophosphate decarboxylase, R-glycerol phosphate dehydrogenase, and triosephosphate isomerase catalyze dissimilar decarboxylation, hydride transfer, and proton transfer reactions, respectively. Each enzyme derives ca. 12 kcal/mol of transition state stabilization from protein interactions with the nonreacting phosphate group, which is larger than the highest 10 kcal/mol transition state stabilization that we have determined for small-molecule catalysis of phosphate diester cleavage in water. Each of these enzymes catalyze the slow reaction of a truncated substrate that lacks the phosphate group, and in each case, the reaction of the truncated substrate is strongly activated by the allosteric binding of the second substrate piece phosphite dianion, HPO32-. We propose a modular design for these enzymes with a classical active site that recognizes the reactive substrate fragment and a separate phosphodianion binding site. The second site is created, in part, by exible protein loops that wrap around the substrate phosphodianion group and bury the substrate in an environment with an optimal local dielectric constant for the catalyzed reaction and with the most favorable positioning of the catalytic side chains. This design is easily generalized to a wide variety of enzyme-catalyzed reactions.
Published on the Web 02/23/2008 www.pubs.acs.org/acr 10.1021/ar7002013 CCC: $40.75 2008 American Chemical Society
Vol. 41, No. 4
April 2008
539-548
ACCOUNTS OF CHEMICAL RESEARCH
539
Phosphate Binding Energy and Catalysis Morrow et al.
Introduction
Linus Pauling noted that catalysis in general, and by enzymes in particular, will result from the development of tight interactions between the catalyst and the transition state for the catalyzed reaction.1 This is shown by the transition state binding energy, GSq (Scheme 1). Catalysis by most enzymes is so efcient that release of products would be strongly rate-determining if they were to bind with the same afnity as the transition state. Consequently, enzymes show a large discrimination and bind their substrates/products much more weakly than the reaction transition state (Scheme 1).2 The Pauling paradigm has a particular appeal when trying to explain catalysis to students. On the other hand, there are issues that must be addressed if this model is to be accepted as a starting framework for explaining enzyme catalysis. These include the following: (1) Recently, Zhang and Houk wrote: While complementarity of the type proposed by Pauling can account for acceleration up to 11 orders of magnitude, most enzymes exceed that prociency.3 They therefore proposed that enzymes achieve over 15 kcal/mol of transition state binding not merely by a concatenation of noncovalent effects but by covalent bond formation between enzyme or cofactor and transition state, involving a change in mechanism from that in aqueous solution.3 (2) There are only small differences in the structure of the substrate/product and the transition state for many enzymecatalyzed reactions. The origin of the large differential binding of these species is generally unknown.2 (3) Enzymes are large molecules that undergo many types of motion, some of which may be coupled to catalysis. Further, catalysis of chemical reactions must ultimately be explained at the level of quantum mechanics. Since neither protein dynamics nor quantum mechanics are central to the Pauling paradigm, more sophisticated models for catalysis might either use this paradigm as a starting point or as a specialized example or, in the most extreme case, discard it entirely. Our goal as experimentalists has been to characterize the mechanisms for catalysis by small metal ion complexes and by larger enzymes where much or all of the catalytic power is derived from the utilization of phosphate binding energy. We summarize here the results of this work and provide an interpretation of our results within the framework of the Pauling model.
540 ACCOUNTS OF CHEMICAL RESEARCH 539-548 April 2008 Vol. 41, No. 4
SCHEME 1
Phosphate Diester Cleavage

The design of catalysts to cleave RNA has considerable intellectual appeal, along with potential to produce RNA cleavage reagents that have practical applications.46 J. R. Morrow and co-workers have shown that lanthanide(III) complexes are efcient catalysts of the cleavage of RNA,7,8 and they have examined cleavage of the 5 cap of mRNA by several metal ion-macrocycle complexes.9 Recent collaborative work between the Morrow and Richard laboratories has focused on characterizing the rate acceleration for catalysis by metal ion complexes and dening the origin of the catalytic rate acceleration. Kinetic Analyses. An examination of the literature suggested to us that the laboratory time expended in the synthesis of novel metal ion catalysts of RNA cleavage often exceeded the time spent in characterizing their kinetics and mechanism of action. In particular, it is difcult to compare the catalytic activity of metal ion complexes prepared in different laboratories, because there is no commonly agreed upon protocol for measuring and reporting the kinetic parameters for catalysis. Our initial goal was to develop such a protocol for reporting kinetic data for metal ion complex-catalyzed phosphate diester cleavage in water at 25 C, in the hope that it might be adopted by other laboratories. We have reported second-order rate constants kMe for the catalyzed cleavage of HpPNP, UpPNP, and UpU to form cyclic
broad range of mono- and dinuclear catalysts are also governed by ionization of a metal-bound water, as shown in Scheme 2.1019 The data in Figure 1 and that for related catalyzed reactions show a good t to eq 1 derived for Scheme 2, which gave values of Ka similar to those determined by potentiometric titration.10
kMe )
kZnKa Ka + [H+]
(1)
FIGURE 1. pH-rate proles for cleavage of HpPNP catalyzed by HO- and Zn(II) complexes in water at 25 C:10 ([) kobsd (s-1) for spontaneous HO--catalyzed cleavage; (9), kMe (M-1 s-1) for cleavage catalyzed by Zn(2)(H2O); (b), kMe (M-1 s-1) for cleavage catalyzed by Zn2(1)(H2O); (1), ratio of kMe for cleavage catalyzed by the dinuclear (kdi) and mononuclear (kmono) Zn(II) complexes.
phosphate diesters. These rate constants are generally determined as the slopes of linear plots of kobsd (s-1) for phosphate diester cleavage against the catalyst concentration. The rate constant kMe has the same units (M-1 s-1) and meaning as kcat/Km for an enzyme-catalyzed reaction. Values of kMe determined at Buffalo and elsewhere can be directly compared in order to provide a simple and meaningful measure of the catalytic activity of different metal ion complexes or of their activity relative to enzyme catalysts. The catalytic effectiveness of a variety of metal ion complexes was then evaluated over a broad range of pH. Figure 1 shows representative pH-rate proles for the Zn2(1)(H2O)and Zn(2)H2O-catalyzed cleavage of HpPNP and for the spon-
Figure 1 shows that the relative activity of Zn2(1)(H2O) and Zn(2)(H2O) as catalysts of the cleavage of HpPNP depends upon whether the observed second-order rate constants kMe are compared at high or low pH. There is only a 12-fold difference in the apparent reactivity of the dinuclear complex and the mononuclear complex at high pH. This difference increases to 290-fold for reactions at low pH because the downward break in catalytic activity is observed at a higher pH for Zn(2)(H2O), as a result of the 1.2 unit higher pKa for ionization of the zinc-bound water at Zn(2)(H2O) than of that at Zn2(1)(H2O).10 The high catalytic activity of Zn2(1)(H2O) is notable because tethering two mononuclear Zn2+ complexes to form a dinuclear complex often causes only a statistical ca. 2-fold increase in catalytic activity.15 The X-ray structure of crystals (grown at pH 6.0) of the perchlorate salt of Zn2(1)(H2O) shows that the two Zn2+ are separated by only 3.66 , due to shielding of the electrostatic interaction between the metal cations by the bridging alkoxide.10 The interactions between the two Zn2+ and the linker alkoxide anion draw the metal cations into a densely charged core, which provides a particularly large electrostatic stabilization of the dianionic transition state for phosphate diester cleavage.10,15
Transition State Binding Energies

Our next goal was to obtain a simple measure of catalytic effectiveness from our kinetic data. The rate accelerations for the catalyzed cleavage of HpPNP, given by eq 2 derived for Scheme 3, were calculated from the relative displacement of the parallel lines for the formally HO--catalyzed cleavage ([, Figure 1) and the HO--dependent metal ion complex catalyzed reactions (b and 9), where Ka is the ionization constant for the metal-bound water and Kw ) 10-14 M2.20 The transition state binding energies of GSq ) -9.6 and - 5.9 kcal/mol for the reactions catalyzed by Zn2(1)(H2O) (Ka ) 107.8 M) and Zn(2)(H2O) (Ka ) 109.2 M) respectively, correspond to (1.1 10 7)-fold and (2.2 104)-fold rate accelerations over the specic-base-catalyzed reaction.18,20 Zn2(1)(H2O) is an extremely impressive small
Vol. 41, No. 4 April 2008 539-548 ACCOUNTS OF CHEMICAL RESEARCH 541
SCHEME 2
taneous specic-base-catalyzed cleavage of HpPNP.10 These proles show that the complex between HpPNP and the fully protonated catalyst is inactive and that this complex is converted to the active form upon loss of a proton. Maximal activity is observed at high pH, where the metal-bound water of the free catalyst is ionized. The pH- and pD-rate proles for catalysis of the cleavage of HpPNP, UpPNP, and UpU by a
SCHEME 3
molecule catalyst. Metal ion complexes with even higher catalytic activity in water21 and in methanol22 have been prepared.
GSq ) GMeq - GHOq ) - RT ln
kZnKa/Kw kHO
(2)
Figure 2 shows the transition state binding energy as the difference in the activation barrier for HO--catalyzed cleavage of an RNA analogue and for the metal ion complex-catalyzed reaction, which is also an HO--dependent reaction (eq 2). The calculation of GSq compresses extensive kinetic data into a single parameter that provides a direct measure for catalytic activity at neutral pH, where the catalyst is largely protonated. We have determined the values of GSq for a wide variety of metal ion complex-catalyzed reactions and used these data to evaluate the effect of changing catalyst and substrate structure on catalytic activity.11,14,16,1820
FIGURE 2. Bar graph that shows the barrier for HO--promoted phosphate diester cleavage in water (GHOq, green bar), the barrier for the metal ion complex-catalyzed hydroxide ion-promoted reaction (GMeq, green bar), and the binding energy for the transition state for the aqueous HO--promoted reaction (GSq).
SCHEME 4
Active Form of Catalyst

The pH dependence (Figure 1) of the uncatalyzed and catalyzed cleavage of HpPNP at pH less than the catalyst pKa shows that under these conditions a proton is lost from the catalyst or substrate on proceeding to the rate-determining transition state. This kinetic analysis cannot distinguish between (a) the loss of proton from the substrate, in which case Zn2(1)(H2O) is the active catalyst and (b) the loss of a proton from the catalyst, in which case the active form of the catalyst is Zn2(1)(HO-), whose concentration approaches a limit at pH > pKa (Scheme 2).10,12 Analysis of inhibition of the Zn2(1)(H2O)-catalyzed cleavage of HpPNP shows that methylphosphate dianion binds to Zn2(1)(H2O) with a 1600-fold higher afnity than does diethylphosphate monoanion.23 This strong and specic binding of the stable dianion resembles the specic binding of the dianionic transition state for cleavage of HpPNP to Zn2(1)(H2O), with an afnity that is much greater than that of the substrate monoanion.10 We concluded that methyl phosphate dianion is a transition state analogue for the Zn2(1)(H2O)-catalyzed cleavage of phosphate diesters.24
Apparent inhibition constants, Ki, for inhibition of Zn2(1)(H2O)-catalyzed cleavage of HpPNP by methyl phosphate dianion approach a limiting small value of 6 10-6 M for formation of a tight complex at low pH, where the catalyst exists mainly in the protonated form Zn2(1)(H2O).23 These data (not shown) were t to the model in Scheme 4 in which the transition state analogue dianion binds to the active protonated catalyst, Zn2(1)(H2O), but not to the ionized catalyst Zn2(1)(HO-). We concluded that Zn2(1)(H2O) is the active catalyst that stabilizes the transition state dianion and that it is converted to the inactive form Zn2(1)(HO-) when the pH is increased above pKa ) 7.8. Deprotonation of Zn2(1)(H2O) may inactivate the catalyst by changing the favorable displacement of the water ligand by the substrate phosphate monoanion to an unfavorable reaction for the displacement of the strongly basic hydroxide ion ligand. A change in solvent from H2O to D2O causes an increase from 7.8 to 8.4 in the pKa of the zinc-bound water at Zn2(1)(L2O) (Scheme 2), but has little effect on the reactivity of
SCHEME 5
CHART 1
isomerization of trioses, where there is additional stabilization of the transition state from a chelate effect (Scheme 6).26
SCHEME 6
Zn2(1)(L2O) toward cleavage of UpPNP. Therefore, there is no primary kinetic solvent deuterium isotope effect on the cleavage reaction that results from movement of a hydron in the rate-determining transition state,12 such as occurs in reactions where there is concerted general base catalysis (GBC, Scheme 5A). We concluded that the phosphate diester cleavage reaction follows the specic-base-catalyzed (SBC) pathway shown in Scheme 5B. We have proposed that the SBC pathway is observed for catalysis by these metal ion complexes because of the dominant role played by electrostatics in stabilization of the transition state for cleavage of RNA analogues by interactions with the metal ion complex catalyst (Scheme 5B).12 In other words, that the covalent-type stabilization gained by placing the Brnsted base at the transition state for the GBC reaction (Scheme 5A) is smaller than the electrostatic stabilization lost upon partial neutralization of negative charge at the now partly protonated O-2.
12
StructureReactivity Effects
We next used the above kinetic protocols to examine the effect of systematic changes in the metal cation, ligand, and substrate structure on the transition state binding energy GSq (Scheme 3) for reactions catalyzed by free metal ions and by metal ion complexes. Ligand Effects. Chart 1 shows transition state binding energies (GSq, Scheme 3) for catalysis of the cleavage of HpPNP by several mononuclear Zn2+ complexes and by hydrated Zn2+.18 By comparison, a value of GSq ) -3.3 kcal/mol was determined for Zn2+-catalyzed deprotonation of acetone,25 where Zn2+ interacts with a monoanionic rather than a dianionic transition state. A value of GSq ) -6.1 kcal/ mol was determined for Zn2+-catalyzed aldose-ketose
Zn2+ alone is a good catalyst of the cleavage of HpPNP (Chart 1). However, it is not possible to obtain large rate constants for this catalyzed cleavage at high pH, because of the sparing solubility of hydrated Zn 2+. Ligands 2 - 5 strongly enhance catalysis by increasing the total concentration of soluble metal cation. The small variation in GSq across Chart 1 suggests a similar origin for the transition state stabilization, which we propose is mainly stabilizing electrostatic interactions between the metal cation and transition state dianion.18 Chart 1 shows that macrocycle ligands either increase or decrease the catalytic activity compared with the case where there is no ligand but that the effect on GSq is no more than 1 kcal/mol. The formation of Zn2+ complexes has the effect of shifting positive charge from Zn2+ to the electron-donor ligand atom so that the weakest complexes will tend to show the highest charge density at Zn2+. The complexes of Zn2+ to macrocycles 4 and 5 are substantially weaker than the complexes to 2 and 3, but the weakly bound Zn2+ at the former complexes shows the greater catalytic activity (Chart 1).18 The higher activity of Zn(4)(H2O) and Zn(5)(H2O) may be due to the larger positive charge density at the more weakly coordinated Zn2+, and the resulting enhancement of stabilization from electrostatic interactions with the dianionic transition state.18 Cation Effects. The second-order rate constants for cleavage of HpPNP catalyzed by Cu2(1)(H2O) (Chart 2) are much smaller than those for catalysis by Zn2(1)(H2O) under the same conditions and are invariant between pH 7 and 10.14 The X-ray crystal structure for Cu2(1)(H2O) shows a bridging alkoxide linker and a Cu(II)-Cu(II) distance of 3.58 , 27 similar to that observed in the crystal structure for Zn 2( 1 )(H 2O). 10 The structure for Zn 2( 1 )(H 2O) shows one hexacoordinated and one pentacoordinated Zn(II),10 while the structure for Cu2(1)(H2O) shows two pentacoordinated
CHART 2
the dimeric complex operate independently in catalyzing the cleavage of HpPNP. Specicity. There are signicant differences in the rate accelerations for the Zn2(1)(H2O)-catalyzed cleavage of RNA analogues and a dinucleoside monophosphate that reect the different specicities of the dinuclear complex Zn2(1)(H2O) for transition state binding (Chart 4).10,11,17,20 The binding site at this catalyst has not been characterized; however, it should not show strong shape complementarity to any of these substrates. We proposed other origins for the range of transition state binding energies shown in Chart 4.
CHART 4
Cu(II). 27 We proposed that the coordination site missing from Cu2(1)(H2O) is essential for the efcient binding and catalysis of the reaction of HpPNP. The larger absolute transition state binding energy of GSq ) 9.8 kcal/mol for cleavage of HpPNP catalyzed by Eu(III) (mainly [Eu(OH2)9]3+)19 in water than for cleavage catalyzed by hydrated Zn2+ in water (GSq ) 6.6 kcal/mol, Chart 1)18 is probably due to the stronger stabilizing electrostatic interaction of a trication than of a dication with the transition state dianion. Chart 3 gives the values of GSq for catalysis of cleavage of HpPNP by mononuclear Eu(III) complexes of 616 and 7,19 and for a dinuclear complex that forms by spontaneous dimerization of [Eu(6)(OH2)2]+ at pH g 7.5.16
CHART 3
(1) The difference between GSq ) -9.6 and -7.2 kcal/mol for catalysis of cleavage of HpPNP, a minimal substrate, and of the more bulky substrate UpPNP, respectively, suggests that close approach of the phosphate diester to the metal cations is required for effective catalysis and that there is steric hindrance to the approach of the larger substrate UpPNP to Zn2(1)(H2O).11 (2) The difference between GSq ) -9.3 and -7.1 kcal/mol for catalysis of cleavage of UpOEt and UpOCH2CCl3, respectively, is a consequence of the different Brnsted parameters lg ) -1.28 and -0.72 for the specic-base-catalyzed and Zn2(1)(H2O)-catalyzed cleavage reactions.17,28 This corThe absolute value of GSq ) 8.6 kcal/mol for catalysis by [Eu(7)(OH2)2]3+ is only 1.2 kcal/mol larger than that observed for the most active mononuclear Zn(II) catalysts (Chart 1). Apparently, the larger number of donor groups to Eu(III) at [Eu(7)(OH2)2]3+ compared with mononuclear Zn(II) catalysts act to attenuate the 3.2 kcal/mol larger transition state stabilization that is observed for catalysis by Eu(III) when all the ligands are water. The 1.5 kcal/mol larger absolute value of GSq for [Eu(7)(OH2)2]3+ than for [Eu(6)(OH2)2]+ shows that there is a small increase in the catalytic efciency with increasing total positive charge at the catalyst available to interact with the transition state dianion. Finally, the similar transition state binding energies for catalysis by [Eu(6)(OH2)2]+ and [Eu2(6)2(OH)(OH2)2]+ provide evidence that the two metal ions in
responds to the neutralization of an effective transition state charge of ca. 0.56 units by interactions between the catalyst and the leaving group anion.29 These data show that there is stabilization of the transition state for cleavage of UpOEt from interaction of the cationic catalyst with both the reacting phosphate core and the strongly basic alkoxy leaving group. (3) The difference between GSq ) -9.3 and -7.2 kcal/mol for catalysis of cleavage of UpU and UpPNP, respectively, is due partly or entirely to transition state stabilization by interaction of the catalyst with the strongly basic alkoxy leaving group at UpU. There may also be a weak nonspecic interaction between the catalyst and the second pyrimidine base at UpU.20
Lessons from Studies on Small Molecule Catalysis

The high catalytic activity of Zn2(1)(H2O) arises from strong stabilizing binding interactions (GSq, Scheme 3) between the dianionic transition state and the densely charged cationic core of Zn2(1)(H2O).10 No concerted general base catalysis of phosphate diester cleavage is observed, presumably because anything gained from such catalysis is offset by an even larger reduction in the electrostatic stabilization of the transition state (Scheme 5). A distinguishing feature of these metal ion complex-catalyzed reactions is their large discrimination between the binding of the substrate phosphate diester monoanion (weak) and of the oxyphosphorane-like transition state dianion30 and the methyl phosphate dianion transition state analogue (strong).23 These results provide evidence that transition state stabilization by mononuclear and dinuclear metal ion complexes is due largely to electrostatic interactions, which are optimal for the dianionic transition state and transition state analogue. In other words, good catalysis of the cleavage of phosphate diesters in water has been obtained relatively easily because of the intrinsically strong stabilizing interactions between densely charged metals and phosphate dianions. The total transition state binding energy GSq can be modied by interactions between the metal ion and its ligands, but these effects are small in comparison with the transition state stabilization observed for catalysis by free metal ions, where the ligands are water (Chart 1).
stereospecic 1,2-hydrogen shift at (R)-glyceraldehyde 3-phosphate (GAP) to give dihydroxyacetone phosphate (DHAP). A single base (Glu-165) catalyzes suprafacial proton transfer through a cis-enediol(ate) intermediate.34 The ratio of secondorder rate constants (kcat/Km)GAP/(kcat/Km)GA ) (2.4 108 M-1 s-1)/(0.34 M-1 s-1) ) 7 108 for the TIM-catalyzed isomerization of GAP and of (R)-glyceraldehyde shows that binding interactions between the enzyme and the phosphodianion group of GAP provide g12 kcal/mol of transition state stabilization.35 This may account for all but 2 kcal/mol of the transition state stabilization for TIM-catalyzed isomerization of GAP.35,36
CHART 5
Enzyme Catalysis
A major difference between small molecule and enzyme catalysts is that the latter have evolved mechanisms for utilization of the binding interactions between the protein and nonreacting portions of the substrate in stabilization of the transition state for the catalyzed reaction.2 The transition state binding energies of up to 10 kcal/mol observed for catalysis of the cleavage of phosphate diesters are due mainly to electrostatic interactions between the catalyst and the phosphate group, which is the reaction center. We have observed even larger transition state stabilizations of ca. 12 kcal/mol from utilization of the binding energy between enzyme catalysts and the nonreacting phosphodianion group of substrates for enzyme-catalyzed proton transfer, hydride transfer, and decarboxylation reactions.3133
Reactions of Triosephosphates
Triosephosphate isomerase (TIM) is a prototypical catalyst of proton transfer at carbon (Chart 5). This enzyme catalyzes the
The value of (kcat/Km)Gly ) 0.26 M-1 s-1 for TIM-catalyzed exchange of deuterium from solvent D2O into the truncated substrate glycolaldehyde is similar to (kcat/Km)GA ) 0.34 M-1 s-1 for isomerization of (R)-glyceraldehyde.31 The deuterium exchange reaction of glycolaldehyde is strongly activated by addition of the second substrate piece phosphite dianion, HPO32-. The data give (kcat/Km)E HPi ) 185 M-1 s-1 for turnover of glycolaldehyde by TIM that is saturated with phosphite dianion, so binding of phosphite dianion to TIM results in a 700-fold rate acceleration of proton transfer from carbon. The two substrate pieces, glycolaldehyde and HPO32-, each bind weakly to TIM, and there is a large chelate effect for binding of the whole substrate GAP (Chart 5).31,37 The binding of HPO32- to free TIM (Kd ) 38 mM) is 700-fold weaker than its binding to the eeting TIM transition state complex (Kdq ) 53 M, Scheme 7). This corresponds to an intrinsic phosphite dianion binding energy of -5.8 kcal/mol (Scheme 7).31 The proton transfer reaction catalyzed by TIM occurs in an active site at which the substrate is sequestered from solvent.38,39 We proposed a model for catalysis by TIM in
SCHEME 7
OMPDC and that complexed with the transition state analogue 6-hydroxyuridine 5 -monophosphate shows that ligand binding results in a large motion to close the active site with the formation of numerous proteinligand contacts, including ve hydrogen bonds to the phosphodianion group. 45 The phosphodianion at OMP, or at any phosphorylated enzymatic substrate, may simply anchor the substrate to the enzyme, or the enzyme conformational change effected by the small remote group may directly assist in the creation of an active site that provides optimal transition state stabilization.
CHART 6
which part of the total intrinsic binding energy of the phosphodianion group of substrate is utilized to drive a protein conformational change that sequesters the substrate at an active site with an apparent dielectric constant substantially lower than that of solvent and with the catalytic groups optimally organized to stabilize the transition state for deprotonation of R-carbonyl carbon.31,40
SCHEME 8
The whole substrate DHAP is reduced by NADH to give L-glycerol 3-phosphate in a reaction catalyzed by R-glycerol phosphate dehydrogenase (Scheme 8).41 The ratio (kcat/Km)DHAP/(kcat/Km)Gly ) (1 106 M-1 s-1)/(0.009 M-1 s-1) ) 1.1 108 for rabbit muscle R-glycerol phosphate dehydrogenase-catalyzed reduction of DHAP and glycolaldehyde gives an intrinsic phosphate binding energy of -11 kcal/mol, which is similar to that for TIM.33 We nd that phosphite dianion is also a powerful activator of enzyme-catalyzed reduction of the truncated neutral substrate glycolaldehyde by NADH.33 X-ray crystallographic analysis of human R-glycerol phosphate dehydrogenase provides evidence that the phosphate binding energy of the substrate is used to drive the closure of a loop over the substrate.41
Orotidine 5-Monophosphate Decarboxylase (OMPDC)

OMPDC is a remarkable enzyme that effects an enormous 1017-fold acceleration of the chemically very difcult decarboxylation of orotidine 5-monophosphate (OMP, Chart 6) to give uridine 5-monophosphate (UMP).42 The enzymatic 43 and nonenzymatic 44 decarboxylation reactions proceed through the vinyl carbanion intermediates. Comparison of the X-ray crystal structures of free yeast
The intrinsic binding energy of the phosphodianion group of OMP, calculated from the ratio of second-order rate constants kcat/Km for the OMPDC-catalyzed reactions of OMP (9.4 106 M-1 s-1)46 and the truncated substrate 1-(-D-erythrofuranosyl)orotic acid (EO, 2.1 10-2 M-1 s-1)32 is -12 kcal/ mol (Chart 6). The weak binding of the EO (Kd 0.1 M) and phosphite dianion (Kd ) 0.14 M) pieces to OMPDC is striking in view of the tight binding of the whole substrate OMP (Km ) 1.6 M).37,46 Decarboxylation of EO is strongly activated by phosphite dianion, with (kcat/Km)E HPi ) 1600 M-1 s-1 for turnover of EO by OMPDC that is saturated with HPO32-, so the binding of this dianion to OMPDC results in an 80000-fold acceleration of decarboxylation.32 The binding of HPO32- to OMPDC (Kd ) 0.14 M) must also be 80000-fold weaker than its binding to the eeting complex of OMPDC with EO in the transition state (Kdq ) 1.8 M, Scheme 7). The total intrinsic binding energy of HPO32- at the transition state complex is therefore -7.8 kcal/mol. This is divided into the small -1.2
kcal/mol binding energy observed in the ground-state complex and an additional -6.6 kcal/mol interaction that develops on proceeding to the transition state for enzyme-catalyzed decarboxylation of EO.2,32
Tina L. Amyes received B.A. and M.A. degrees in natural sciences and her Ph.D. degree in chemistry (1986) from the University of Cambridge (England). She is currently an Adjunct Associate Professor at the University at Buffalo, SUNY. John P. Richard received a B.S. degree in biochemistry and his Ph.D. degree in chemistry (1979) from The Ohio State University. He is currently Professor of Chemistry at the University at Buffalo, SUNY.
FOOTNOTES * E-mail: jrichard@chem.buffalo.edu. REFERENCES 1 Pauling, L. The nature of forces between large molecules of biological interest. Nature 1948, 161, 707709. 2 Jencks, W. P. Binding energy, specicity and enzymic catalysis: The circe effect. Adv. Enzymol. Relat. Areas Mol. Biol. 1975, 43, 219410. 3 Zhang, X.; Houk, K. N. Why enzymes are procient catalysts: Beyond the Pauling paradigm. Acc. Chem. Res. 2005, 38, 379385. 4 Morrow, J.; Iranzo, O. Synthetic metallonucleases for RNA cleavage. Curr. Opin. Chem. Biol. 2004, 8, 192200. 5 Williams, N. H.; Takashi, B.; Wall, M.; Chin, J. Structure and nuclease activity of simple dinuclear metal complexes: quantitative dissection of the role of metal ions. Acc. Chem. Res. 1999, 32, 485493. 6 Blasko, A.; Bruice, T. C. Recent studies of nucleophilic, general-acid and metal ion catalysis of phosphate diester hydrolysis. Acc. Chem. Res. 1999, 32, 475484. 7 Chin, K. O. A.; Morrow, J. R. RNA cleavage and phosphate diester transesterication by encapsulated lanthanide ions: Traversing the lanthanide series with lanthanum(III), europium(III), and lutetium(III) complexes of 1,4,7,10-tetrakis(2hydroxyalkyl)-1,4,7,10-tetraazacyclododecane. Inorg. Chem. 1994, 33, 50365041. 8 Morrow, J. R.; Buttrey, L. A.; Shelton, V. M.; Berback, K. A. Efcient catalytic cleavage of RNA by lanthanide(III) macrocyclic complexes: Toward synthetic nucleases for in vivo applications. J. Am. Chem. Soc. 1992, 114, 19031905. 9 Baker, B. F.; Khalili, H.; Wei, N.; Morrow, J. R. Cleavage of the 5 cap structure of mRNA by a europium(III) macrocyclic complex with pendant alcohol group. J. Am. Chem. Soc. 1997, 119, 87498755. 10 Iranzo, O.; Kovalevsky, A. Y.; Morrow, J. R.; Richard, J. P. Physical and kinetic analysis of the cooperative role of metal ions in catalysis of phosphodiester cleavage by a dinuclear Zn(II) complex. J. Am. Chem. Soc. 2003, 125, 19881993. 11 Yang, M.-Y.; Richard, J. P.; Morrow, J. R. Substrate specicity for catalysis of phosphodiester cleavage by a dinuclear Zn(II) complex. Chem. Commun. 2003, 28322833. 12 Yang, M.-Y.; Iranzo, O.; Richard, J. P.; Morrow, J. R. Solvent deuterium isotope effects on phosphodiester cleavage catalyzed by an extraordinarily active Zn(II) complex. J. Am. Chem. Soc. 2005, 127, 10641065. 13 Feng, G.; Mareque-Rivas, J. C.; Williams, N. H. Comparing a mononuclear Zn(II) complex with hydrogen bond donors with a dinuclear Zn(II) complex for catalysing phosphate ester cleavage. Chem. Commun. 2006, 18451847. 14 Iranzo, O.; Richard, J. P.; Morrow, J. R. Structure-activity studies on the cleavage of an RNA analogue by a potent dinuclear metal ion catalyst. The effect of changing the metal ion. Inorg. Chem. 2004, 43, 17431750. 15 Iranzo, O.; Elmer, T.; Richard, J. P.; Morrow, J. R. Cooperativity between metal ions in the cleavage of phosphate diesters and RNA by dinuclear Zn(II) catalysts. Inorg. Chem. 2003, 42, 77377746. 16 Farquhar, E.; Richard, J. P.; Morrow, J. R. Formation and stability of mononuclear and dinuclear Eu(III) complexes and their catalytic reactivity toward cleavage of an RNA analogue. Inorg. Chem. 2007, 46, 71697177. 17 Yang, M.-Y. Ph.D. Thesis, University at Buffalo, 2007. 18 Mathews, R. A.; Rossiter, C. S.; Morrow, J. R.; Richard, J. P. A minimalist approach to understanding the efciency of mononuclear Zn(II) complexes as catalysts of cleavage of an RNA analogue. Dalton Trans. 2007, 38043811. 19 Nwe, K.; Richard, J. P.; Morrow, J. R. Direct excitation luminescence spectroscopy of Eu(III) complexes of 1,4,7-tris(carbamoylmethyl)-1,4,7,10- tetraazacyclododecane derivatives and kinetic studies of their catalytic cleavage of an RNA analog. Dalton Trans. 2007, 51715178. 20 ODonoghue, A.; Pyun, S. Y.; Yang, M.-Y.; Morrow, J. R.; Richard, J. P. Substrate specicity of an active dinuclear complex for cleavage of RNA analogues and a dinucleoside. J. Am. Chem. Soc. 2006, 128, 16151621.
The Pauling Paradigm

Our studies have shown that the interactions between nonreacting phosphodianion fragments of substrate and the protein provide an 1112 kcal/mol stabilization of the transition states for proton transfer, hydride transfer, and decarboxylation reactions. Therefore, enzymes that catalyze the reactions of small phosphorylated substrates such as GAP and DHAP have a potential transition state binding energy of -12 kcal/mol. The medium-sized phosphorylated substrate OMP interacts with OMPDC via its phosphodianion, ribose sugar ring, and nucleic acid base portions and exhibits a transition state binding energy far in excess of -15 kcal/mol that was proposed as the limit on noncovalent enzyme catalysis.3,42 Pyridoxal 5-phosphate (PLP) provides impressive covalent catalysis of the deprotonation of R-amino acids to form R-amino carbanions.47 However, the large rate acceleration for proton transfer obtained by recruitment of this cofactor can be also obtained by protein catalysts, such as proline racemase. This enzyme provides a 19 kcal/mol stabilization of the transition state for deprotonation of enzyme-bound proline by interaction with the protein catalyst.48 Our proposal that pure protein catalysis of deprotonation of amino acids results from the binding of the amino acid substrate at a nonpolar active site that favors formation of a carbanion zwitterion49,50 has been supported by X-ray crystallographic and computational studies.51,52 We are generally skeptical of arguments that cofactors such as PLP have evolved to do what is impossible for protein catalysts, because of the great diversity of protein-catalyzed reactions. On the other hand, cofactor catalysis is often simpler and more general than protein catalysis, and these factors favor the recruitment of cofactors in enzymatic catalysis. We acknowledge the NSF (Grant CHE0415356) and the NIH (Grant GM39754) for support of our work, and we thank our co-workers named in the references for their many contributions to our research.
BIOGRAPHICAL INFORMATION Janet R. Morrow received a B.S. degree in chemistry from the University of California, Santa Barbara, and her Ph.D. degree in chemistry (1985) from the University of North Carolina, Chapel Hill. She is currently Professor of Chemistry at the University at Buffalo, SUNY.
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21 Feng, G.; Mareque-Rivas, J. C.; Torres Martin de Rossales, R.; Williams, N. H. A highly reactive mononuclear Zn(II) complex for phosphodiester cleavage. J. Am. Chem. Soc. 2005, 127, 1347013471. 22 Neverov, A. A.; Lu, Z. L.; Maxwell, C. I.; Mohamed, M. F.; White, C. J.; Tsang, J. S. W.; Brown, R. S. Combination of a dinuclear Zn2+ complex and a medium effect exerts a 1012-fold rate enhancement of cleavage of an RNA and DNA model system. J. Am. Chem. Soc. 2006, 128, 1639816405. 23 Yang, M.-Y.; Morrow, J. R.; Richard, J. P. A transition state analog for phosphate diester cleavage catalyzed by a small enzyme-like metal ion complex. Bioorg. Chem. 2007, 35, 366374. 24 Wolfenden, R. Transition state analogues for enzyme catalysis. Nature 1969, 223, 704705. 25 Crugeiras, J.; Richard, J. P. A comparison of the electrophilic reactivities of Zn2+ and acetic acid as catalysts of enolization: imperatives for enzymatic catalysis of proton transfer at carbon. J. Am. Chem. Soc. 2004, 126, 51645173. 26 Nagorski, R. W.; Richard, J. P. Mechanistic imperatives for aldose-ketose isomerization in water: specic-, general base- and metal ion-catalyzed isomerization of glyceraldehyde with proton and hydride transfer. J. Am. Chem. Soc. 2001, 123, 794802. 27 Fry, F. H.; Moubaraki, B.; Murray, K. S.; Spiccia, L.; Warren, M.; Skelton, B. W.; White, A. H. Asymmetry in endogenously bridged binuclear copper(II) and zinc(II) complexes formed by 1,2-bis[1,4,7-triazacyclonon-1-yl]-propan-2-ol. Dalton Trans. 2003, 866871. 28 Kosonen, M.; Youseti-Salakdeh, E.; Stromberg, R.; Lnnberg, H. Mutual isomerization of uridine 2- and 3-alkylphosphates and cleavage to a 2,3-cyclic phosphate: the effect of the alkyl group on the hydronium- and hydroxide-ioncatalyzed reactions. J. Chem. Soc., Perkin Trans. 2 1997, 26612666. 29 Williams, A. Electronic charge and transition state structure in solution. Adv. Phys. Org. Chem. 1992, 27, 155. 30 Perreault, D. M.; Anslyn, E. V. Unifying the current data on the mechanism of cleavage-transesterication of RNA. Angew. Chem., Int. Ed. Engl. 1997, 36, 432 450. 31 Amyes, T. L.; Richard, J. P. Enzymatic catalysis of proton transfer at carbon: Activation of triosephosphate isomerase by phosphite dianion. Biochemistry 2007, 46, 58415854. 32 Amyes, T. L.; Richard, J. P.; Tait, J. J. Activation of orotidine 5-monophosphate decarboxylase by phosphite dianion: the whole substrate is the sum of two parts. J. Am. Chem. Soc. 2005, 127, 1570815709. 33 Tsang, W.-Y.; Amyes, T. L.; Richard, J. P. A Substrate in Pieces: Allosteric Activation of Glycerol 3-Phosphate Dehydrogenase (NAD+) by Phosphite Dianon, Biochemistry, submitted for publication. 34 Knowles, J. R.; Albery, W. J. Perfection in enzyme catalysis: The energetics of triosephosphate isomerase. Acc. Chem. Res. 1977, 10, 105111. 35 Amyes, T. L.; ODonoghue, A. C.; Richard, J. P. Contribution of phosphate intrinsic binding energy to the enzymatic rate acceleration for triosephosphate isomerase. J. Am. Chem. Soc. 2001, 123, 1132511326. 36 Richard, J. P. Acid-base catalysis of the elimination and isomerization reactions of triose phosphates. J. Am. Chem. Soc. 1984, 106, 49264936. 37 Jencks, W. P. On the attribution and additivity of binding energies. Proc. Natl. Acad. Sci. U.S.A. 1981, 78, 40464050.
38 ODonoghue, A. C.; Amyes, T. L.; Richard, J. P. Hydron transfer catalyzed by triosephosphate isomerase. Products of isomerization of (R)-glyceraldehyde 3phosphate in D2O. Biochemistry 2005, 44, 26102621. 39 ODonoghue, A. C.; Amyes, T. L.; Richard, J. P. Hydron transfer catalyzed by triosephosphate isomerase. Products of isomerization of dihydroxyacetone phosphate in D2O. Biochemistry 2005, 44, 26222631. 40 Sampson, N. S.; Knowles, J. R. Segmental motion in catalysis: Investigation of a hydrogen bond critical for loop closure in the reaction of triosephosphate isomerase. Biochemistry 1992, 31, 84888494. 41 Ou, X.; Ji, C.; Han, X.; Zhao, X.; Li, X.; Mao, Y.; Wong, L.-L.; Bartlam, M.; Rao, Z. Crystal structure of human glycerol 3-phosphate dehydrogenase (GPDI). J. Mol. Biol. 2006, 357, 858869. 42 Radzicka, A.; Wolfenden, R. A procient enzyme. Science 1995, 267, 9093. 43 Toth, K.; Amyes, T. L.; Wood, B. M.; Chan, K.; Gerlt, J. A.; Richard, J. P. Product deuterium isotope effect for orotidine 5-monophosphate decarboxylase: Evidence for the existence of a short-lived carbanion intermediate. J. Am. Chem. Soc. 2007, 129, 1294612947. 44 Sievers, A.; Wolfenden, R. Equilibrium of formation of the 6-carbanion of UMP, a potential intermediate in the action of OMP decarboxylase. J. Am. Chem. Soc. 2002, 124, 1398613987. 45 Miller, B. G.; Hassell, A. M.; Wolfenden, R.; Milburn, M. V.; Short, S. A. Anatomy of a procient enzyme: The structure of orotidine 5-monophosphate decarboxylase in the presence and absence of a potential transition state analog. Proc. Natl. Acad. Sci. U.S.A. 2000, 97, 20112016. 46 Porter, D. J. T.; Short, S. A. Yeast orotidine-5-phosphate decarboxylase: Steadystate and pre-steady-state analysis of the kinetic mechanism of substrate decarboxylation. Biochemistry 2000, 39, 1178811800. 47 Toth, K.; Richard, J. P. Covalent catalysis by pyridoxal: Evaluation of the effect of the cofactor on the carbon acidity of glycine. J. Am. Chem. Soc. 2007, 129, 3013 3021. 48 Williams, G.; Maziarz, E. P.; Amyes, T. L.; Wood, T. D.; Richard, J. P. Formation and stability of the enolates of N-protonated proline methyl ester and proline zwitterion in aqueous solution: a nonenzymatic model for the rst step in the racemization of proline catalyzed by proline racemase. Biochemistry 2003, 42, 83548361. 49 Rios, A.; Amyes, T. L.; Richard, J. P. Formation and stability of organic zwitterions in aqueous solution: Enolates of the amino acid glycine and its derivatives. J. Am. Chem. Soc. 2000, 122, 93739385. 50 Richard, J. P.; Amyes, T. L. On the importance of being zwitterionic: Enzymic catalysis of decarboxylation and deprotonation of cationic carbon. Bioorg. Chem. 2004, 32, 354366. 51 Puig, E.; Garcia-Viloca, M.; Gonzalez-Lafont, A.; Lluch, J. M. On the ionization state of the substrate in the active site of glutamate racemase. A QM/MM study about the importance of being zwitterionic. J. Phys. Chem. A 2006, 110, 717725. 52 Pillai, B.; Cherney, M. M.; Diaper, C. M.; Sutherland, A.; Blanchard, J. S.; Vereras, J. C.; James, M. N. G. Structural insights into stereochemical inversion by diaminopimilate epimerase: an antibacterial drug target. Proc. Natl. Acad. Sci. U.S.A. 2006, 103, 86688673.
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Copyright 2008 by the American Chemical Society
Volume 47, Number 16
April 22, 2008
Accelerated Publications A Substrate in Pieces: Allosteric Activation of Glycerol 3-Phosphate Dehydrogenase (NAD+) by Phosphite Dianion
Wing-Yin Tsang, Tina L. Amyes, and John P. Richard*
Department of Chemistry, UniVersity at Buffalo, State UniVersity of New York, Buffalo, New York 14260-3000 ReceiVed January 30, 2008; ReVised Manuscript ReceiVed March 5, 2008
The ratio of the second-order rate constants for reduction of dihydroxyacetone phosphate (DHAP) and of the neutral truncated substrate glycolaldehyde (GLY) by glycerol 3-phosphate dehydrogenase (NAD+, GPDH) saturated with NADH is (1.0 106 M-1 s-1)/(8.7 10-3 M-1 s-1) ) 1.1 108, which was used to calculate an intrinsic phosphate binding energy of at least 11.0 kcal/mol. Phosphite dianion binds very weakly to GPDH (Kd > 0.1 M), but the bound dianion strongly activates GLY toward enzyme-catalyzed reduction by NADH. Thus, the large intrinsic phosphite binding energy is expressed only at the transition state for the GPDH-catalyzed reaction. The ratio of rate constants for the phosphiteactivated and the unactivated GPDH-catalyzed reduction of GLY by NADH is (4300 M-2 s-1)/(8.7 10-3 M-1 s-1) ) 5 105 M-1, which was used to calculate an intrinsic phosphite binding energy of -7.7 kcal/mol for the association of phosphite dianion with the transition state complex for the GPDH-catalyzed reduction of GLY. Phosphite dianion has now been shown to activate bound substrates for enzymecatalyzed proton transfer, decarboxylation, hydride transfer, and phosphoryl transfer reactions. Structural data provide strong evidence that enzymic activation by the binding of phosphite dianion occurs at a modular active site featuring (1) a binding pocket complementary to the reactive substrate fragment which contains all the active site residues needed to catalyze the reaction of the substrate piece or of the whole substrate and (2) a phosphate/phosphite dianion binding pocket that is completed by the movement of exible protein loop(s) to surround the nonreacting oxydianion. We propose that loop motion and associated protein conformational changes that accompany the binding of phosphite dianion and/or phosphodianion substrates lead to encapsulation of the substrate and/or its pieces in the protein interior, and to placement of the active site residues in positions where they provide optimal stabilization of the transition state for the catalyzed reaction.
Enzymes are protein catalysts that effect rate accelerations much larger than those observed for small molecule catalysts. The larger rate accelerations for enzymes are a consequence of the greater binding afnities of enzymes for their reaction transition states (1). Enzyme catalysis is so efcient that the release of products would be intolerably slow if they were
This work was supported by Grant GM39754 from the National Institutes of Health. * To whom correspondence should be addressed. Telephone: (716) 645-6800, ext. 2194. Fax: (716) 645-6963. E-mail: jrichard@ chem.buffalo.edu.
ABSTRACT:
to bind with the same afnity as the transition state. For example, the ca. 30 kcal/mol transition state intrinsic binding energy estimated for the enzymatic decarboxylation of orotidine 5-monophosphate (OMP)1 catalyzed by orotidine
Abbreviations: OMP, orotidine 5-monophosphate; OMPDC, orotidine 5-monophosphate decarboxylase; GAP, (R)-glyceraldehyde 3-phosphate; TIM, triosephosphate isomerase; GPDH, glycerol 3-phosphate dehydrogenase (NAD+); DHAP, dihydroxyacetone phosphate; NADH, nicotinamide adenine dinucleotide, reduced form; R-GP, L-glycerol 3-phosphate; GLY, glycolaldehyde; BSA, bovine serum albumin; DTT, D,L-dithiothreitol; TEA, triethanolamine; NMR, nuclear magnetic resonance.
1
10.1021/bi8001743 CCC: $40.75 2008 American Chemical Society Published on Web 04/01/2008
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Biochemistry, Vol. 47, No. 16, 2008

Scheme 2
Accelerated Publication
Scheme 1
5-monophosphate decarboxylase (OMPDC) (2) is even larger than the 20 kcal/mol binding energy associated with the effectively irreversible binding of biotin to avidin (3). Consequently, to avoid this free energy trap, enzymes generally bind their substrates and/or products much more weakly than the reaction transition state. It has been proposed that enzymes often produce large rate accelerations through the utilization of binding interactions between the protein and nonreacting portions of the substrate for transition state stabilization (4, 5). However, the mechanism(s) by which enzyme catalysts selectively turn on these binding interactions at the transition state for their catalyzed reactions is not generally known, and this question is often omitted from discussions of the origin of the rate acceleration for enzyme catalysts. The binding energy of the remote nonreacting phosphodianion groups of OMP and (R)-glyceraldehyde 3-phosphate (GAP) has been shown to provide a ca. 12 kcal/mol stabilization of the transition states for OMPDC-catalyzed decarboxylation (6) and proton transfer catalyzed by triosephosphate isomerase (TIM) (7), respectively. Moreover, the small substrate piece phosphite dianion provides strong allosteric-like activation of OMPDC-catalyzed decarboxylation (6) and TIM-catalyzed deprotonation (8) of a second truncated substrate piece from which the phosphodianion group has been excised. The latter results show that a substantial fraction of the intrinsic phosphate binding energy is expressed as transition state stabilization even in the absence of a covalent connection between the substrate pieces. Phosphite dianion binds weakly to both TIM (Kd ) 38 mM) (8) and OMPDC (Kd ) 140 mM) (6) so that the large intrinsic phosphite binding energy is expressed only at the respective transition states for these enzyme-catalyzed reactions. X-ray crystallographic analyses suggest that both TIM and OMPDC utilize the binding energy of the substrate phosphodianion group to drive the thermodynamically unfavorable closure of a mobile protein loop(s) to cover the bound phosphodianion (912). We have proposed (8) that this loop closure sequesters the substrate from bulk solvent and results in formation of a local microenvironment in which there is optimal stabilization of the transition state for the enzymatic reaction (13, 14). We examine here whether the functional equivalence of GAP and the two-part substrate [glycolaldehyde + phosphite] (Scheme 1) for proton transfer catalyzed by TIM might also be observed for other types of enzymatic reactions. Glycerol 3-phosphate dehydrogenase (NAD+-linked, EC 1.1.1.8, GPDH) is used in the assay for TIM-catalyzed isomerization of GAP to couple formation of the product
dihydroxyacetone phosphate (DHAP) to its reduction by NADH to give L-glycerol 3-phosphate [R-GP (Scheme 2)] (15). We report here that GPDH also catalyzes the very slow reduction of the neutral minimal substrate glycolaldehyde (GLY) by NADH and that the separate binding of the second substrate piece phosphite dianion strongly activates GPDH for catalysis of the reduction of GLY by NADH. We conclude that the substrate pieces [glycolaldehyde + phosphite] are able to effectively substitute for the whole substrate GAP or DHAP in enzyme-catalyzed proton transfer and hydride transfer reactions, respectively. MATERIALS AND METHODS Materials. Glycerol 3-phosphate dehydrogenase (lyophilized powder, 75 units/mg) from rabbit muscle was purchased from USB Corp. Bovine serum albumin, fraction V (BSA), was from Roche. Dihydroxyacetone phosphate (lithium salt), NADH (disodium salt), glycolaldehyde dimer, triethanolamine hydrochloride, D,L-dithiothreitol (DTT), and sodium deuteroxide (40 wt %, 99.9% D) were from Sigma-Aldrich. Deuterium oxide (99.9% D) was from Cambridge Isotope Laboratories. Sodium phosphate (dibasic) was from Fluka, and sodium phosphite (dibasic, pentahydrate) was from Riedel-de Haen (Fluka). Water was from a Milli-Q Academic purication system. All other chemicals were reagent grade or better and were used without further purication. Preparation of Solutions. The solution pH was determined as described previously (8). Stock solutions of NADH were prepared by dissolving NADH (disodium salt) in water and were stored at 4 C. The concentration of NADH was determined spectrophotometrically at 340 nm using an of 6220 M-1 cm-1. Stock solutions of DHAP were prepared on the day of the experiment by dissolving the lithium salt in water and adjusting the pH to 7.5 using 1 M NaOH and were stored on ice. The concentration of DHAP was determined spectrophotometrically at 340 nm as the concentration of NADH oxidized in a reaction catalyzed by GPDH. GPDH (2 or 20 mg/mL) in 20 mM triethanolamine (TEA) buffer (pH 7.5, 32% free base) was dialyzed exhaustively against the same buffer at 5 C. The concentration of GPDH was determined from the absorbance at 280 nm using an A of 5.15 for a 1% solution of GPDH (16) and a subunit molecular mass of 37500 Da. Stock solutions of glycolaldehyde (100 mM monomer) were prepared by dissolving glycolaldehyde dimer in water and storing the solution for at least 3 days at room temperature to allow for breakdown of the dimer to the monomer (8). An apparent pKa of 6.38 for phosphite monoanion at 25 C and an I of 0.12 (NaCl) was determined as the pH of a 30 mM solution of the sodium salt at a 1:1 monoanion:dianion ratio. Enzyme Assays. Dilutions of the dialyzed stock solutions of 2 mg/mL enzyme (used for assays of GPDH-catalyzed reduction of DHAP) and 20 mg/mL enzyme (used for assays of GPDH-catalyzed reduction of GLY) were made in 20 mM TEA (pH 7.5, 32% free base) containing 10 mM DTT.
Accelerated Publication All enzyme assays were conducted at 25 C and an I of 0.12 (NaCl) in a volume of 1 mL. Initial velocities of oxidation of NADH (e5% reaction) were calculated from the changes in absorbance at 340 nm using a of 6220 M-1 cm-1. The standard assay mixture for GPDH activity contained 100 mM TEA (pH 7.5), 1.2 mM DHAP, 0.20 mM NADH, 0.1 mg/mL BSA, 50 M DTT, and ca. 1 nM GPDH at an I of 0.12 (NaCl). Reactions were initiated by making a 200-fold dilution of the diluted stock enzyme containing 10 mM DTT into the reaction mixture. The same procedure was used to determine the velocity of GPDH-catalyzed reduction over a range of initial concentrations of DHAP, except that the buffer was 20 mM TEA (pH 7.5). Assay mixtures for the GPDH-catalyzed reduction of GLY in the absence of phosphite contained 10 mM TEA (pH 7.5), 20 mM GLY (total), 0.20 mM NADH, and up to ca. 0.2 mM GPDH at an I of 0.12 (NaCl). Assay mixtures for the GPDH-catalyzed reduction of GLY in the presence of phosphite contained 10 mM TEA (pH 7.5), 2-60 mM GLY (total), 0.20 mM NADH, up to 32 mM phosphite dianion, and 0.6 M GPDH at an I of 0.12 (NaCl). 1H NMR Analyses. 1H NMR spectra at 500 MHz were recorded in D2O at 25 C using a Varian Unity Inova 500 spectrometer as described previously (8). The product of the GPDH-catalyzed reduction of GLY by NADH in a reaction mixture containing 10 mM TEA (pD 8.0), 31 mM sodium phosphite (93% dianion), 5 mM GLY (total), and 7 mM NADH in D2O was identied by 1H NMR spectroscopy. The reaction was initiated by the addition of 380 L of GPDH in buffered D2O (pD 8.0, 20 mM TEA) to give a total volume of 1 mL and a nal enzyme concentration of 11 M. A second 1H NMR spectrum, recorded after reaction for 12 h, showed the disappearance of ca. 80% of the starting GLY and the appearance of a singlet at 3.53 ppm, which was shown to be due to the methylene protons of a corresponding amount of ethylene glycol by the addition of an authentic standard. The fraction of GLY present in the free carbonyl form in H2O buffered by 10 mM TEA (pH 7.5) at 25 C and an I of 0.12 (NaCl) (1 - fhyd) was determined by 1H NMR spectroscopy using a coaxial inner tube containing D2O and suppression of the water peak. A value for 1 - fhyd of 0.06 ( 0.003 was obtained by comparison of the integrated areas of the signals due to the C2 protons of the free carbonyl and hydrated forms of GLY, which is the same as a value for 1 - fhyd of 0.06 determined previously for the hydration of GLY in D2O at pD 7.0, 25 C, and an I of 0.10 (8). RESULTS Initial velocities, Vi, of the reduction of DHAP by NADH catalyzed by GPDH at pH 7.5 (10 mM TEA), 25 C, and an I of 0.12 (NaCl) were determined by monitoring the decrease in absorbance at 340 nm. Figure 1A shows the dependence of Vi (M s-1) on the total concentration of DHAP in the presence of 0.10 mM (2) or 0.20 mM (b) NADH and 1.1 nM GPDH. The solid line shows the t of the data to the MichaelisMenten equation with a (Km)app value of 0.24 mM and a Vmax value of 1.42 10-7 M s-1. The latter was used to calculate a kcat value of 130 s-1, and a Km value of 0.13 mM for the reactive free carbonyl form of DHAP was calculated using a value for 1 - fhyd of 0.55 for the fraction
Biochemistry, Vol. 47, No. 16, 2008 4577
FIGURE 1: (A) Dependence of the initial velocity, Vi, of the reduction of DHAP by NADH catalyzed by GPDH (1.1 nM) on the total concentration of DHAP in the presence of 0.10 mM (2) or 0.20 mM (b) NADH at pH 7.5, 25 C, and an I of 0.12 (NaCl). (B) Dependence of the initial velocity, Vi, of the reduction of GLY (20 mM total, 1.2 mM free carbonyl form) by NADH (0.20 mM) on the concentration of GPDH at pH 7.5, 25 C, and an I of 0.12 (NaCl).
Table 1: Kinetic Parameters for Reduction of Dihydroxyacetone Phosphate and Glycolaldehyde Catalyzed by GPDH and for Activation by Phosphite Dianiona Kmb kcat substrate activator (mM) (s-1) kcat/Km (M-1 s-1) K dc (M) kcat/KiaKb (M-2 s-1)
DHAPd 0.13 130 1.0 106 GLYe (8.7 ( 0.5) 10-3 GLYf HPO32- 8 ( 1 >0.1 4300 ( 700
a At pH 7.5 (10 mM TEA buffer), 25 C, and an I of 0.12 (NaCl). Values of Km refer to the free carbonyl form of DHAP or GLY. c Dissociation constant for the phosphite dianion activator. d Kinetic parameters determined from data shown in Figure 1A. e Kinetic parameters determined from data shown in Figure 1B. f Kinetic parameters determined from data shown in Figure 2. b
Scheme 3
of DHAP present in the free carbonyl form (Scheme 3) (17). These data (Table 1) are in agreement with the published kinetic parameters for GPDH (18). There is a ca. 50% decrease in the activity of GPDH (60 M) during an 8 h incubation at pH 7.5 (10 mM TEA) in the presence of 1.2 mM GLY (free carbonyl form, 20 mM total GLY) and 0.20 mM NADH at 25 C and an I of 0.12 (NaCl). Figure 1B shows the dependence of the initial velocity Vi (M s-1) of the reduction of GLY under these
4578

Scheme 4
quaternary complex E NADH GLY HPO32- are each much smaller than the total concentration of GPDH over the range of HPO32- concentrations examined. The data were therefore t to eq 1, derived for a reaction mechanism in which there is no signicant formation of the E NADH HPO32- complex (Scheme 4). The linear correlations in Figure 2A show that [HPO32-] , Kb, and the slopes of these correlations correspond to apparent second-order rate constants (kcat/Km)app (M-1 s-1) for the phosphite-activated GPDH-catalyzed reduction of GLY by NADH (eq 2). Vi [E] ) kcat[GLY][HPO32-] [GLY][HPO32-] + Kb[GLY] + KiaKb (kcat Kb)[GLY] Kia + [GLY] (1) (2)
(kcat Km)app )
FIGURE 2: (A) Dependence of Vi/[E] for the reduction of GLY by NADH (0.20 mM) catalyzed by GPDH (0.6 M) on the concentration of exogenous phosphite dianion at pH 7.5, 25 C, and an I of 0.12 (NaCl). Concentrations of GLY (free carbonyl form) were (3) 0.12, (4) 0.30, (0) 0.60, (1) 1.2, (b) 1.8, (2) 2.4, ([) 3.0, and (9) 3.6 mM. (B) Dependence of the apparent second-order rate constant (kcat/Km)app for the phosphite-activated GPDH-catalyzed reduction of GLY by NADH (0.20 mM) on the concentration of GLY at pH 7.5, 25 C, and an I of 0.12 (NaCl), calculated as the slopes of the correlations in panel A.
conditions on the concentration of GPDH, determined from the decrease in absorbance at 340 nm (0.01-0.02 OD unit) observed at early reaction times (e60 min) where the velocity is constant. The loss of activity of GPDH during this time, determined by a periodic standard assay, was <10%. The slope of the correlation in Figure 1B gives the apparent rstorder rate constant for the enzymatic reduction of GLY as (1.04 ( 0.06) 10-5 s-1.2 The second-order rate constant for enzyme-catalyzed reduction of GLY (Scheme 3) can then be calculated with the relationship (kcat/Km)GLY ) (1.04 10-5 s-1)/[GLY] ) (8.7 ( 0.5) 10-3 M-1 s-1, where [GLY] (1.2 mM) is the concentration of GLY present in the reactive free carbonyl form. Figure 2A shows that there is a linear dependence of Vi/ [E] (s-1) for the reduction of various concentrations of GLY by NADH (0.20 mM) catalyzed by GPDH (0.6 M) on the concentration of exogenous phosphite dianion at pH 7.5 (10 mM TEA), 25 C, and an I of 0.12 (NaCl). The addition of phosphite dianion results in very large increases in Vi for these GPDH-catalyzed reactions, and 1H NMR analysis conrmed that the product of the reduction of GLY by NADH in the presence of phosphite dianion is ethylene glycol (Scheme 4). The failure to observe curvature in the correlations shown in Figure 2A requires that phosphite dianion bind very weakly to GPDH so that the concentrations of both the ternary complex E NADH HPO32- and the
Quoted errors are standard deviations obtained from the linear or nonlinear least-squares t of the data. Errors in calculated quantities were computed using standard equations for the propagation of error.
2
Figure 2B shows the dependence of (kcat/Km)app, determined as the slopes of the correlations in Figure 2A, on the concentration of the free carbonyl form of glycolaldehyde. The solid line shows the t of the data to eq 2, which gave a Kia value of 8 ( 1 mM for binding of GLY and a kcat/Kb value of 35 ( 4 M-1 s-1 (Scheme 4). The slope of the correlation in Figure 2B at low concentrations of GLY corresponds to the third-order rate constant kcat/KiaKb (4300 ( 700 M-2 s-1) for the phosphite-activated GPDH-catalyzed reduction of GLY by NADH. The kinetic parameters determined in this work are summarized in Table 1. The velocity of the GPDH-catalyzed reduction of DHAP by NADH (0.20 mM) at [DHAP] ) Km, pH 7.5 (10 mM TEA), 25 C, and an I of 0.12 (NaCl) was found to decrease by 15% upon the inclusion of 33 mM phosphite dianion, while 35 mM sulfate dianion causes a somewhat larger 36% decrease in the velocity under the same reaction conditions. These small changes in velocity show that both phosphite and sulfate dianions bind only weakly to GPDH. The evaluation of Ki binding constants for these dianions is complicated by uncertainties about whether there are also specic salt effects on the kinetic parameters for the GPDHcatalyzed reaction. DISCUSSION The cofactor NAD+ is a competitive product inhibitor of the GPDH-catalyzed reduction of DHAP when NADH is the varied substrate but a noncompetitive inhibitor when DHAP is the varied substrate (19). The sugar L-glycerol 3-phosphate is a noncompetitive product inhibitor of the GPDH-catalyzed reduction of DHAP when either NADH or DHAP is the varied substrate, but the inhibition with respect to NADH changes to uncompetitive when the reaction is carried out in the presence of saturating concentrations of DHAP (19). These results provide strong evidence for an ordered mechanism, with NADH binding rst followed by DHAP (18, 19). A similar ordered mechanism is assumed to hold for reduction of the two-part substrate [glycolaldehyde + phosphite] by NADH, with the cofactor binding rst, followed by binding of the substrate pieces GLY and
Biochemistry, Vol. 47, No. 16, 2008 4579
Table 2: Comparison of the Intrinsic Phosphate Binding Energies and the Observed and Total Intrinsic Phosphite Binding Energies for Enzyme-Catalyzed Decarboxylation, Proton Transfer, and Hydride Transfer Reactionsa
a Binding energies are dened to be negative when the binding is thermodynamically favorable. b Second-order rate constant for turnover of the whole phosphorylated natural substrate by the free enzyme. c Second-order rate constant for turnover of the neutral truncated substrate lacking a phosphodianion group by the free enzyme. d Calculated from the rate constant ratio (kcat/Km)SPi/(kcat/Km)S. e Third-order rate constant for enzyme-catalyzed reaction of the neutral truncated substrate activated by phosphite dianion. f Energy for binding of phosphite dianion to the free enzyme (top number) and to the transition state complex E Sq (bottom number). g Data from ref 6. h Data from ref 8. i Data from this work.
phosphite dianion (Scheme 4). A Michaelis constant of 6.3 M for NADH has been reported for the GPDH-catalyzed reduction of DHAP at pH 7.8 (19). The essentially identical dependence of the initial velocity of GPDH-catalyzed reduction on DHAP concentration in the presence of 0.10 or 0.20 mM NADH at pH 7.5 (Figure 1A) shows that under our reaction conditions GPDH is completely saturated by the lower concentration of NADH. Studies of the reduction of GLY and of the phosphite-activated reduction of GLY catalyzed by GPDH were carried out in the presence of 0.20 mM NADH so that the concentration of the E NADH complex is given by the total concentration of enzyme. Reaction of the Substrate in Pieces. The MichaelisMenten plots for the reduction of DHAP catalyzed by GPDH at 0.10 or 0.20 mM NADH and pH 7.5 (Figure 1A) give a Km of 0.13 mM for the free carbonyl form of DHAP and a kcat/Km of 1.0 106 M-1 s-1 as the second-order rate constant for reduction of DHAP by the E NADH complex. By comparison, a value for (kcat/Km)GLY of (8.7 ( 0.5) 10-3 M-1 s-1 was determined for reduction of the truncated substrate GLY by the E NADH complex (Figure 1B). We conclude that the substrate phosphodianion group activates DHAP toward GPDH-catalyzed reduction by NADH by at least 108-fold. This likely underestimates the phosphodianion group activation because the aldehyde group of GLY is intrinsically more reactive toward a nucleophilic hydride donor than the keto group of DHAP. These data show that the enzymatic transition state for hydride transfer from enzyme-bound NADH to DHAP is stabilized by at least 11 kcal/mol by interactions between GPDH and the nonreacting phosphodianion group of the substrate (Table 2). The GPDH-catalyzed reduction of GLY by NADH is strongly activated by the second substrate piece phosphite dianion. Figure 2A shows that plots of Vi/[E] (s-1) for reduction of various concentrations of GLY by NADH (0.20 mM) at pH 7.5 [I ) 0.12 (NaCl)] against HPO32- concentration are linear up to at least 33 mM phosphite dianion. The same concentration of phosphite dianion results in only a 15% reduction in the initial velocity of GPDH-catalyzed
Scheme 5
reduction of DHAP by NADH when [DHAP] ) Km. We conclude that phosphite dianion binds only weakly to GPDH (Kd > 0.1 M for dissociation of the E HPO32- complex) but that this dianion provides an impressive activation of reduction of the second substrate piece GLY by NADH catalyzed by GPDH. The slopes of the linear correlations in Figure 2A correspond to apparent second-order rate constants (kcat/Km)app for the phosphite-activated reduction of GLY catalyzed by the E NADH complex. Figure 2B shows that these apparent second-order rate constants appear to approach a limiting value for reactions at high concentrations of the hydride acceptor GLY. The t of the data in Figure 2B to eq 2 (derived for Scheme 4) gave a kcat/KiaKb of 4300 ( 700 M-2 s-1 as the third-order rate constant for reaction of the two substrate pieces GLY and HPO32- with the E NADH complex to form ethylene glycol and NAD+. Scheme 5 shows that the intrinsic afnity of HPO32- for the GPDHtransition state complex E Sq, dened as a Kdq of 2 M for the E HPO32- Sq complex, can be calculated as the ratio of the second-order rate constant for unactivated enzymecatalyzed reduction of GLY, (kcat/Km)GLY, and the third-order rate constant for reaction of the substrate pieces, kcat/KiaKb, according to eq 3. Kdq ) (kcat Km)GLY 0.0087 M-1 s-1 ) ) 2.0 M kcat KiaKb 4300 M-2 s-1 (3)
The observation of linear plots in Figure 2A for the phosphite-activated enzyme-catalyzed reduction of GLY by NADH shows that there is no detectable accumulation of
4580
FIGURE 3: (A) Free energy diagram for turnover of the truncated neutral substrate GLY (S) by the binary GPDH NADH complex (E) and for the same reaction activated by the binding of exogenous phosphite dianion (HPO32-). Activation free energies were calculated using the Eyring equation at 298 K. The weakly bound Michaelis complex E HPO32- is not shown, because the stability of this complex is not dened by our experiments. (B) Free energy diagram illustrating our proposed model for activation of E toward reduction of GLY by the binding of HPO32-. Binding energies are dened to be negative when the binding is thermodynamically favorable. The difference between the total intrinsic phosphite binding energy of -7.7 kcal/mol and the observed binding energy x of greater than -1.4 kcal/mol for binding of HPO32- to the inactive open enzyme EO to give the active closed enzyme EC HPO32- is the binding energy that is used to convert EO to EC. The observed Gq value of 20.2 kcal/mol for turnover of GLY (panel A) has been partitioned into the Gq for reduction of GLY by EC and the Gconf for the unfavorable conformational change that converts EO to EC.
the E HPO32- complex at e33 mM HPO32-. Therefore, the binding of phosphite dianion to the free enzyme is not shown in Scheme 5, because the stability of the E HPO32- complex relative to the free enzyme is not dened by our experiments. Scheme 5 is represented graphically in Figure 3A. The large activation barrier of ca. 20 kcal/mol for the direct bimolecular reduction of the truncated neutral substrate GLY by the E NADH complex was calculated from the secondorder rate constant (kcat/Km)GLY [(8.7 ( 0.5) 10-3 M-1 s-1 (Table 1)] using the Eyring equation at 298 K. The smaller activation barrier for the phosphite-activated reduction of GLY was calculated from the third-order rate constant kcat/ KiaKb [4300 ( 700 M-2 s-1 (Table 1)]. The 7.7 kcal/mol larger activation barrier for reduction of GLY catalyzed by the E NADH complex compared to that for its phosphiteactivated reduction is equal to the stabilization associated with the binding of phosphite dianion to the transition state complex E Sq to give the complex E HPO32- Sq, for which Kdq ) 2 M. Mobile Loops and Catalysis. X-ray crystallographic analysis of GPDH from Leishmania mexicana shows that the GPDH NAD+ DHAP ternary complex adopts a closed form relative to the free, unliganded, enzyme (20). It has been proposed that the formation of stabilizing interactions between the phosphodianion group of bound DHAP and amino acid side chains at mobile protein elements provides the major driving force for closure of the protein pocket (20, 21). This is analogous to the closure of exible loops around the phosphodianion group of bound intermediate analogues at TIM (11, 12, 22) and at OMPDC (9, 23). In these cases, loop closure and the formation of interactions with the substrate phosphodianion group were proposed to activate the substrates GAP/DHAP and OMP for the respective enzyme-catalyzed proton transfer (8) and decarboxylation (6) reactions. Figure 3B is a free energy diagram that illustrates one physical mechanism for activation of GPDH toward reduction of glycolaldehyde by the binding of exogenous phosphite dianion. In this model, we propose that a large fraction of the intrinsic binding energy of -7.7 kcal/mol for the
phosphite dianion activator is utilized to drive a thermodynamically unfavorable change in the conformation of the enzyme from an inactive open form, EO (E ) E NADH), to an active loop-closed form, EC. The obserVed binding of phosphite dianion to the free enzyme is therefore only weakly favorable, with a binding energy x > -1.4 kcal/mol, because the total intrinsic phosphite binding energy is largely offset by the unfavorable free energy associated with the conformational changes that accompany binding of this substrate piece (Gconf ) 7.7 + x kcal/mol). We also propose that a large fraction of the intrinsic binding energy of the transition state for reduction of the neutral substrate piece GLY, Sq, is utilized to drive the same loop closure and conformational change. The full intrinsic binding energy of HPO32- or of the transition state Sq is then observed only when these species bind to the loop-closed forms of GPDH, EC Sq or EC HPO32-, respectively. We propose that the large selectivity of phosphite dianion for binding to EC rather than to EO results from the formation of stabilizing interactions between this oxydianion and amino acid side chains at mobile protein elements that move to form the loop-closed protein, and that very similar interactions are formed between the protein and the phosphodianion group of the whole substrate DHAP. The proposed large selectivity of the transition state for binding to EC would require that loop closure result in formation of a local environment that is more favorable for transformation of the bound substrate to the transition state for hydride transfer than that at the open enzyme EO. We are able only to speculate about these changes, because the imperatives for enzymatic catalysis of hydride transfer from NADH have not been well dened. Loop closure that sequesters the substrate from aqueous solvent at a nonpolar active site should have the effect of increasing the extent of transition state stabilization available from hydrogen bonding and ion pairing interactions between the protein and the bound ligand (24). For example, the magnitude of the stabilizing electrostatic interactions between enzymic hydrogen bond donors and the partial negative charge that develops at the carbonyl oxygen in
Accelerated Publication the transition state for hydride transfer from NADH to glycolaldehyde will increase with a decrease in the effective dielectric constant of the reaction medium (25). It is possible that loop closure when substrate binds to GPDH results in a decrease in the separation between the hydride donor and the hydride acceptor at the Michaelis complex. This would narrow the width of the potential energy barrier that separates reactants and products, and it could favor quantum mechanical tunneling through this barrier (26). Finally, loop closure may have the effect of ordering the catalytic groups in the active site to provide optimal transition state stabilization by minimizing the entropic barrier to the enzyme-catalyzed reaction (13). How Do Enzymes Work? Glucose 1-phosphate and glucose 6-phosphate are rapidly phosphorylated by the phosphoserine of phosphoglucomutase to form glucose 1,6-diphosphate. In 1976, B. Ray showed that enzyme-catalyzed phosphorylation of xylose by this enzyme is very slow, but that this phosphoryl transfer reaction is strongly activated by binding of the second substrate piece phosphite dianion (27). It has taken 30 years to generalize these results from studies of an enzyme-catalyzed phosphoryl group transfer reaction to enzyme-catalyzed proton transfer (8), hydride transfer (this work), and decarboxylation reactions (6). Our work on three different enzymes that catalyze mechanistically diverse heterolytic reactions has shown that these catalysts have achieved strikingly similar efciencies in their utilization of the intrinsic binding energy of the substrate phosphodianion group in catalysis (Table 2). (1) OMPDC, TIM, and GPDH exhibit similar 108-109 -fold lower activities for catalysis of the reaction of a truncated substrate that lacks a phosphodianion group, (kcat/ Km)S, than for catalysis of the reaction of the whole substrate, (kcat/Km)SPi. These results suggest that an important outcome of the biological phosphorylation of sugars is to provide enzyme catalysts with an electrostatic handle from which they may obtain ca. 12 kcal/mol of intrinsic binding energy. (2) Each of these very slow enzyme-catalyzed reactions of the appropriate truncated neutral substrate piece is strongly activated by the separate binding of exogenous phosphite dianion. (3) In each case, the second-order rate constant (kcat/Km)SPi for reaction of the whole substrate is larger than the thirdorder rate constant for the phosphite-activated reaction of the truncated substrate piece [4300-12000 M-2 s-1 (see Table 2)]. The difference between the second-order rate constant for reaction of the whole substrate and the thirdorder rate constant for reaction of the pieces reects the large entropic advantage of the unimolecular reaction of the whole substrate, compared with the bimolecular reaction of the pieces (28). (4) The individual substrate pieces exhibit low afnities for the formation of productive Michaelis complexes. However, tethering the two pieces gives a whole substrate that exhibits an enhanced binding afnity due to the chelate effect (28). (5) In each case, binding of the substrate phosphodianion group drives the movement of mobile protein loop(s) to close the active site and cover the phosphate group, which effectively buries the reactive substrate fragment in the
Biochemistry, Vol. 47, No. 16, 2008 4581 protein interior. The binding of phosphite dianion presumably drives a similar conformational change (8). Apparently, these diverse proteins have adopted a common modular design to effect very efcient enzymatic catalysis from utilization of the intrinsic binding energy of the nonreacting phosphate fragment of the substrate. This design features (a) an active site that recognizes the reactive substrate fragment and which contains all of the catalytic amino acid side chains needed to execute the chemistry of catalyzed reaction and (b) a neighboring site for a second nonreactive substrate piece (for example, phosphite dianion), which is completed by the movement of exible protein loops over this piece. This loop movement encapsulates the reactive substrate piece within the protein where the local dielectric constant is smaller than that of water, where the catalytic side chains are positioned to provide optimal transition state stabilization, and where other electrostatic factors may also favor transition state stabilization. In recent years, there have been, at best, small incremental advances in the de novo rational design of synthetic small molecule catalysts or of high molecular mass protein or nucleic acid catalysts. We suggest that the recognition and understanding of this modular design element in enzymes that have evolved to effectively utilize intrinsic phosphate binding energy may guide the efforts of chemical biologists interested in the de novo design of proteins with enzymelike catalytic activities. REFERENCES
1. Pauling, L. (1948) The nature of forces between large molecules of biological interest. Nature 161, 707709. 2. Radzicka, A., and Wolfenden, R. (1995) A procient enzyme. Science 267, 9093. 3. Green, N. M. (1966) Thermodynamics of the binding of biotin and some analogues by avidin. Biochem. J. 101, 774780. 4. Jencks, W. P. (1975) Binding Energy, Specicity and Enzymic Catalysis: The Circe Effect. AdV. Enzymol. Relat. Areas Mol. Biol. 43, 219410. 5. Jencks, W. P. (1987) Economics of enzyme catalysis. Cold Spring Harbor Symp. Quant. Biol. 52, 6573. 6. Amyes, T. L., Richard, J. P., and Tait, J. J. (2005) Activation of orotidine 5-monophosphate decarboxylase by phosphite dianion: The whole substrate is the sum of two parts. J. Am. Chem. Soc. 127, 1570815709. 7. Amyes, T. L., ODonoghue, A. C., and Richard, J. P. (2001) Contribution of phosphate intrinsic binding energy to the enzymatic rate acceleration for triosephosphate isomerase. J. Am. Chem. Soc. 123, 1132511326. 8. Amyes, T. L., and Richard, J. P. (2007) Enzymatic catalysis of proton transfer at carbon: Activation of triosephosphate isomerase by phosphite dianion. Biochemistry 46, 58415854. 9. Miller, B. G., Hassell, A. M., Wolfenden, R., Milburn, M. V., and Short, S. A. (2000) Anatomy of a procient enzyme: The structure of orotidine 5-monophosphate decarboxylase in the presence and absence of a potential transition state analog. Proc. Natl. Acad. Sci. U.S.A. 97, 20112016. 10. Miller, B. G., Snider, M. J., Short, S. A., and Wolfenden, R. (2000) Contribution of enzyme-phosphoribosyl contacts to catalysis by orotidine 5-phosphate decarboxylase. Biochemistry 39, 81138118. 11. Lolis, E., and Petsko, G. A. (1990) Crystallographic analysis of the complex between triosephosphate isomerase and 2-phosphoglycolate at 2.5- resolution: Implications for catalysis. Biochemistry 29, 66196625. 12. Davenport, R. C., Bash, P. A., Seaton, B. A., Karplus, M., Petsko, G. A., and Ringe, D. (1991) Structure of the triosephosphate isomerase-phosphoglycolohydroxamate complex: An analog of the intermediate on the reaction pathway. Biochemistry 30, 58215826.
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13. Cannon, W. R., and Benkovic, S. J. (1998) Solvation, reorganization energy, and biological catalysis. J. Biol. Chem. 273, 2625726260. 14. Warshel, A. (1998) Electrostatic Origin of the Catalytic Power of Enzymes and the Role of Preorganized Active Sites. J. Biol. Chem. 273, 2703527038. 15. Plaut, B., and Knowles, J. R. (1972) pH-Dependence of the triose phosphate isomerase reaction. Biochem. J. 129, 311320. 16. Bentley, P., Dickinson, F. M., and Jones, I. G. (1973) Purication and properties of rabbit muscle L-glycerol 3-phosphate dehydrogenase. Biochem. J. 135, 853859. 17. Reynolds, S. J., Yates, D. W., and Pogson, C. I. (1971) Dihydroxyacetone phosphate. Its structure and reactivity with R-glycerolphosphate dehdyrogenase, aldolase and triose phosphate isomerase and some possible metabolic implications. Biochem. J. 122, 285297. 18. Bentley, P., and Dickinson, F. M. (1974) A study of the kinetics and mechanism of rabbit muscle L-glycerol 3-phosphate dehydrogenase. Biochem. J. 143, 1927. 19. Black, W. J. (1966) Kinetic studies on the mechanism of cytoplasmic L-R-glycerophosphate dehydrogenase of rabbit skeletal muscle. Can. J. Biochem. Phys. 44, 13011317. 20. Suresh, S., Turley, S., Opperdoes, F. R., Michels, P. A. M., and Hol, W. G. H. (2000) A potential target enzyme for trypanocidal drugs revealed by the crystal structure of NAD-dependent glycerol3-phosphate dehydrogenase from Leishmania mexicana. Structure 8, 541552.

Carbanion in Water

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Carbanion in Water

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Acc. Chem. Res.

2001, 34, 981-988

chemical reactivity is of interest to both organic chemists and biochemists.

Carbocations and Carbanions in Water Richard et al.

(1) (2) (3)

kaz ) 5 109 M-1 s-1 pKR ) -log KR ) log(kH/ks)

982 ACCOUNTS OF CHEMICAL RESEARCH / VOL. 34, NO. 12, 2001

Carbocations and Carbanions in Water Richard et al.

pKa ) pKw + log

pKa ) pKBH + log

Carbocations and Carbanions in Water Richard et al.

Kinetic and Thermodynamic Substituent Effects

Origin of and Changes in Intrinsic Barriers

Carbocations and Carbanions in Water Richard et al.

Carbocations and Carbanions in Water Richard et al.

Absolute and Relative Magnitudes of Intrinsic Barriers to Organic Reactions

Carbocations and Carbanions in Water Richard et al.

Carbocations and Carbanions in Water Richard et al.

988 ACCOUNTS OF CHEMICAL RESEARCH / VOL. 34, NO. 12, 2001

Phosphate Binding Energy and Catalysis by Small and Large Molecules

Vol. 41, No. 4

ACCOUNTS OF CHEMICAL RESEARCH

Phosphate Binding Energy and Catalysis Morrow et al.

Phosphate Diester Cleavage

Phosphate Binding Energy and Catalysis Morrow et al.

Transition State Binding Energies

Phosphate Binding Energy and Catalysis Morrow et al.

GSq ) GMeq - GHOq ) - RT ln

Active Form of Catalyst

Phosphate Binding Energy and Catalysis Morrow et al.

Phosphate Binding Energy and Catalysis Morrow et al.

Phosphate Binding Energy and Catalysis Morrow et al.

Lessons from Studies on Small Molecule Catalysis

Phosphate Binding Energy and Catalysis Morrow et al.

Orotidine 5-Monophosphate Decarboxylase (OMPDC)

Phosphate Binding Energy and Catalysis Morrow et al.

The Pauling Paradigm

Vol. 41, No. 4

ACCOUNTS OF CHEMICAL RESEARCH

Phosphate Binding Energy and Catalysis Morrow et al.

ACCOUNTS OF CHEMICAL RESEARCH

Vol. 41, No. 4

Copyright 2008 by the American Chemical Society

Volume 47, Number 16

April 22, 2008

Biochemistry, Vol. 47, No. 16, 2008

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Biochemistry, Vol. 47, No. 16, 2008

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Biochemistry, Vol. 47, No. 16, 2008

Biochemistry, Vol. 47, No. 16, 2008

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