THE JOURNAL Vol. 249, No.

6, hue

OF BIOLOGICAL CHEMISTRY of March 25, PP. 1848-1856,

in U.S.A.

1974

Printed

Action linked

of Magnesium Ion on Diphosphopyridine Isocitrate Dehydrogenase from Bovine

OF THE FORMS OF THE SUBSTRATE
(Received for publication, GERHARD W. E. PLAUT, VERN L. SCHRAMM, AND TADASHI

NucleotideHeart
OF THE REACTION*

CHARACTERIZATION

AND

THE MODIFIER

November AOGAICHI

22, 1972, and in revised form, July 6, 1973)

From the Department of Biochemistry, Temple University School of Medicine, Philadelphia, Pennsylvania 19160

SUMMARY The activity of DPN-linked isocitrate dehydrogenase is dependent on the concentration of magnesium n-isocitrate (MgI-) under conditions where the concentrations of free Mg2+ and isocitrate approach zero and saturation, respectively. These results can be interpreted in terms of magnesium isocitrate as the active substrate, however the present data cannot rule out the possibility of free Mg2f and isocitrate as the substrates if the reaction mechanism is rapid equilibrium ordered with free Mg*+ being the first reactant. In the absence of ADP and in presence of 0.33 mM DPN+, &o.~ of magnesium DL-isocitrate has been found to be 0.2, 0.4, 0.5, and 1.0 mM at pH 6.65, 7.2, 7.4, and 7.75, respectively, with values of Hill slopes (n) near 2. Excess free Mg2+ causes apparent inhibition which is competitive with respect to magnesium isocitrate, the proposed substrate of the reaction. The inhibition by Mg*+ is much more severe at pH 6.65 than at pH 7.2 and above, e.g. the inhibition constant for Mg2+ is 40. to 80.fold larger at pH 7.2 than at pH 6.65. In contrast to the results which are consistent with magnesium isocitrate as the substrate, free ADP3- is the modifier of the enzyme and MgADPis inactive. At pH 7.2 and 7.4, increasing concentrations of ADP3lead to decreasing values of So.s for magnesium isocitrate accompanied by a decline of Hill slopes from n = 2 to near unity; these changes are less pronounced at pH 6.65. The values of V,,, are the same in the absence and presence of ADP at each pH tested; however, V,,, at pH 6.65 is about one-half of that at pH 7.2 and above. There is an interdependence in the interaction of the enzyme with substrate and modifier. Thus, at pH 7.2 and 7.4, the values of K, for ADP3- decline with increasing concentrations of magnesium isocitrate; however, the Hill slopes for ADP3are not influenced by substrate concentration and remain constant at m = 1 between pH 6.65 and 7.75. A reaction model has been proposed in which initial binding of substrate to the enzyme at one site leads to a conformational change and subsequent binding of magnesium isocitrate at an additional site which is catalytically active.

DPN-dependent isocitrate dehydrogenase from animal tissues (EC 1.1.1.41) catalyzing the reaction n-three-Isocitrate + DPN+ or-ketoglutarate + CO2 + DPNH + H+ has been shown to require Mg2+, Mn+, or Co2+ for activity (l-8). Mn*+ is a more effective activator than Mg*+, exhibiting a lower apparent K, and a somewhat higher Vmax; however, severe inhibition has been noted at higher concentrations of Mn2f (8, 9). With the enzyme from liver the apparent K, of Mg2+ was lowered by increased isocitrate and decreased further by the positive modifier ADP. The question of whether or not a complex of the divalent metal ion with isocitrate is the true substrate for the enzyme was not resolved in the earlier work (8). In recent systematic studies of the role of Mn2+ with a purified enzyme from porcine heart, Cohen and Colman (9) have proposed that dibasic isocitrate is the active form of the substrate of the enzyme with an apparent K, (approximately 30 PM) independent of Mn2+ concentration and pH. Inhibition by substrate, reported at pH 6 but not at pH 7 and 8, was attributed to a complex of manganous dibasic isocitrate. Activation by a number of chelating agents (GDP, UDP, and citrate) was observed (9) and the effect explained as due to removal of Mn2+ from the isocitrate complex resulting in an increase in the active species of isocitrate. In accord with earlier observations (2, 4, 6) these investigators found that ADP lowers the apparent K, of total isocitrate; but they also reported that ADP lowers the K, of dibasic isocitrate. They propose that activation by ADP is caused by direct modification of the enzyme, and by chelation of Mn2+ which leads to increased free dibasic isocitrate. Thus, MS+ would function as a negative regulator; controlling the supply of the substrate (free dibasic isocitrate) and the inhibitor (isocitrate chelate). Nevertheless, Cohen and Colman (9) have confirmed previous observations (l-8) for the absolute requirement for activity of divalent metal ions, although the mechanism of interaction between Mn2+ and enzyme was not elucidated. * This work was supported in part by Grant AM 15404 from the National Institute of Arthritis and Metabolic Diseases, National Institutes of Health.

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1849 Certain
heart differ

of the observations
from previous work

with
with

the enzyme from porcine

the dehydrogenase from

of activity was obother animal tissues. Thus, only inhibition tained with a number of nucleoside di- and triphosphates (other than ADP) with enzymes from bovine and rat heart (2, 4), Ehrlich ascites tumor (6) and liver (S), and the effect has been

attributed partly to chelation of divalent metal ions. With ATP, inhibition competitive with isocitrate occurs at a lower concentration than with other nucleotides (2) ; while it appears to be accompanied by binding of divalent metal ions (4) the
kinetic effect may be due to the specific interaction of enzyme

and ATP which has been shown to occur without added divalent metal ions (10). Citrate is a positive modifier of the enzyme from yeast (ll), Neurosporu (12), and certain other microbial
sources, and is reported by Cohen and Colman (9) to activate

the concentrations indicated in the text, tables, and figures. The concentrations of ADP and DPN+ were determined spectrophotometrically at 260 nm (16) and the magnesium content of stock solutions was assayed by atomic absorption spectrometry.4 Reaction mixtures containing all components except enzyme in a final volume of 1 ml in silica cuvettes of l-cm light path were brought to temperature equilibrium at 25 in a thermostated copper block cuvette holder. Reactions were started by the addition of 5 to 10 ~1 of diluted enzyme solution. Initial reaction rates were determined at 25 by following the reduction of DPN+ spectrophotometrically at 340 nm in a Gilford model 240 spectrophotometer operated in the 0 to 0.2 absorbance range. The spectrophotometer output was recorded with a Honeywell model 190 recorder at a chart speed of 6 to 12 inches per min.

Stock solutions

of the enzyme containing

1.2 mg of protein

per

the enzyme from hog heart; however, with the hog liver enzyme (8). Furthermore, it is difficult to explain
Cohen and Colman (9) protection by

this was not observed with the mechanism

the combination

of

of mag-

nesium (or Mn*) and isocitrate against inhibition by low concentrations of mercurials (3, 8) or by 5,5-dithiobis(2-nitrobenzoate) (13), not obtained with either isocitrate or divalent
metal ion alone.

ml were stored at -90 in a solution containing 10% glycerol, 5 mM sodium phosphate (pH 7.2), and 0.1 mM dithiothreitol. For a days experiment enzyme stock solutions were diluted 1:5 or 1:lO into a solution containing 100 mM sodium Hepes at pH 7.2, 0.1 mM of 1,3-dimercapto-2-propanol, and 764 mM Li.$JOa. Under these conditions no activity was lost in the diluted sample for 21 hours at 0. Velocities are expressed as change in absorbance at 340 nm per min; the values are comparable between experiments since they were calculated in all cases on the basis of the same amount of enzyme protein (1.2 fig) per ml of reaction mixture. Calculations-Grzybowski et al. (17) have reported the stability constants of magnesium isocitrate complexes as

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Therefore, a further clarification of the role of divalent metal ions became desirable. In the studies presented here the role of Mg*+ was investigated. This has the advantage that Mg*+ is
less inhibitory under certain than Mn2+, conditions. an inhibition Evidence which can be irreversible will be presented that and is

K1=

[MgH1l
[Mg*l[HI+]

= 0.027 mM-l

consistent with magnesium isocitrate acting as the true substrate, free ADP is active, and MgADPis inactive as a modifier, and free Mg2+ is not required for activity.
PROCEDURE

[MgI-1 Kz = ~Mg~1~13-l

= 0.521 mM-

where MgHI
and

= magnesium

II-isocitrate;

HI constants

= H-isocitratez-; of isocitric acid

dissocia-

13- = isocitratea-.

EXPERIMENTAL

According

to the acid dissociation

Materials The following chemicals were purchased from the commercial sources indicated. Inorganic salts were analytical reagent grade from Mallinckrodt Chemical Works. 1,3-Dimercapto-2-propanol was from Chemical Procurement Co. ADP, DPN+, EDTA, Hepes,2 Pipes, DL-isocitric la&one, and n-isocitric acid (monopotassium salt) were from Sigma. DL-ISOCitrate lactone was recrystallized by dissolving 1 g in 25 ml of ethyl n-butyrate under reflux followed by treatment with 50 mg of Norit A and filtration of the hot solution. The colorless

(pK,l = 3.02, pK,2 = 4.28, pK,3 = 5.75) and the acid tion constant for magnesium H-isocitrate (17)
K c D&HI1 = [H+lDkI-I

= 3.44 x lo-2

rnM

Isocitrate should be almost fully ionized above pH 7 (i.e. [I] % [I]), and magnesium H-isocitrate formation should be negligible. Therefore, the equilibrium can be described essentially by the used in this communication, constant Kf. With the terminology the equilibrium is described by (1) where MI = (magnesium isocitrate)complex; M = free Mga+; Z = free isocitrate. Above pH 7, the value of KI is assumed to be 0.521 mM-1; however, at pH 6.65, the concentration of H-isocitrate becomes significant and an apparent equilibrium constant of 0.463 rnM-1, calculated from Kz and pK,3 has been used to estimate the concentration of magnesium isocitrate. The concentrations of magnesium isocitrate can be calculated from total magnesium (M!Z) and total isocitrate (IT) by substitution into Equation 1 IMZI

crystals formed overnight

at room temperature

were collected by

filtration, washed with a small quantity of ethyl butyrate, and dried in a vacuum for 3 hours at 95. Recrystallized DL-isocitric lactone was recovered in 75 to 80% yield with m.p. 162. Before use, isocitric lactone was hydrolyzed in alkali as described previously (14) and then adjusted with HCl at 25 to the exact pH used in the incubation mixture. DPN-linked isocitrate dehydrogenase from bovine heart,3

which was prepared by the method of Giorgio et al. (15), showed a

single band in polyacrylamide gel disc electrophoresis and had a specific activity of 28,000 nmoles of DPN+ reduced per mg of protein per min at 25 under the conditions of assay reported previously (14).

Methods
Assay-All incubation mixtures contained 0.33 mM DPN+ and 167 mM sodium Henes at DH 7.2 to 7.75 or 167 mM sodium Pines buffer at pH 6.65. * Isocitrate, ADP, and MgSO4 were added- at 1 C. Fan (1971) unpublished observations. 2 The abbreviations used are: Hepes, N-2-hydroxyethylpiperazinc-N-2-ethanesulfonic acid ; Pipes, piperazine-N,N-bis(2ethanesulfonic acid). 3 These enzyme preparations were made available to us by Dr. C. Fan. and solution

([MI] - [MZI)([ZT]
of the resulting

- [MI])

= Kr

quadratic

equation

KAWI
[MI] The =

+ [MT]) + 1 f d(KAZT] f [MT])

+ 1Y - 4K,*[MTl[ZTl
and free for isocitrate making

(3)

2Kr
concentrations to thank of free Dr. M. Mg2+ can then these de-

4 We wish terminations.

C. Scrutton

1850
be calculated from the relationships [Ml = IMTI [MI] ~0, in which case:

V
The maximal

00)
concentration required (u)) is given by the for one-half relationship:

and [ZI = IZTI - [MI]

For calculation of concentration of the magnesium ADP complex (MgADP-) the stability constants of OSullivan and Perrin (18) were used with values of KA of 2.3, 2.4, and 4.1 rnrv-1 at pH 7.2, 7.4, and 7.75, respectively. The stability constant5 at pH 6.65 was estimated to be 1.4 mM0. The concentrations of free Mg*+ were calculated from total magnesium, total isocitrate and total ADP (AT) according to the relationship of Equation 4, which was solved by use of the computer and the Newton-Raphson iteration technique.

K,,, for
increase

ADP (ADP in velocity

Km = KD.
Calculated values of maximal velocities obtained with the aid of computer fits of the data were used in the construction of Hill plots. Details underlying these calculations are described in the text. All experiments were done in the presence of a constant concentration of DPN+ (0.33 mM). Under the standard assay conditions used to determine the activity of the enzyme (i.e. pH 7.2 and Mn*+ as activator (14)), Km of DPN+ is approximately 0.08 mM (13), and the velocity with 0.33 mM DPN+ is about 80% of

[Ml8 +

&

+ j& + [ATI + UT1 - [MT]

[Ml* [Ml (41

V msx.
RESULTS

[ITI - [MT1 + MT1 - [MT] + K*] KA KI

Initial

Velocity

as Function

A!ELO -- -Concentrations of were then calculated MgADP(MA) and from the relations magnesium isocitrate

When experiments were done with magnesium to total DL-isocitrate,

of Total Isocitrate and Mgzfdifferent fixed ratios of total the results shown in Fig. 1A
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fMzl

[MIUTIK,

1 + KIM

(6)

and concentrations of free ADP (A) and free isocitrate were calculated from the conservation equations6 Since the binding of Mg*+ to DPN+ is relatively weak and the concentration of DPN+ is low it has not been considered here in the distribution of MgZ+ among the various forms. Analysis of Data-The nature of initial velocity curves was determined by graphical analysis and then analyzed by fitting to the appropriate computer programs developed by Cleland (19) using a CDC 6400 digital computer. Data which gave linear double reciprocal plots of initial velocity against substrate concentration were fitted to Equation 7.

FREE

OL-ISOCITRATE,mM

(7)
Data which conformed fitted to Equation 8, Results. to linear with the competitive exceptions inhibition discussed were under

Plots which were rectangular hyperbolas, the origin in plots of ZI against ADP, I

but did not pass through were fitted to Equation 9.

ADP\

v = vo

(9)
Ix)
2.0 MASNESIUY 4.0 CL-ISOClfRAT2,mW 6.0

where ADP.

vg is the initial The maximal

velocity velocity

of the reaction in the absence will be given by setting ADP

of -+

6 OSullivan and Perrin (18) reported that KA = 2.4 rnM-1 at pH 7.4. They estimated that KA = 2.0 rnM-r at pH 7.0 by consideration of the equilibrium HADP+ ti H+ + ADP5-, pK = 6.65. The interpolated value of KA at pH 6.65 was calculated from the same assumptions. li V. L. Schramm (1972) unpublished.

FIG. 1. Changes in substrate concentrations at fixed ratios of total isocitrate (IT) and total Mg*+ (MT) at pH 7.2. Substrate concentrations were varied by addition of solutions with [total Mga+] to [total isocitrate] fixed at the ratios: 0.5, 0 ; 1.0, X ; 2.0, a. A, velocity X10 as a function of free nn-isocitrate concentration; B, velocity as a function of magnesium nn-isocitrate; C, Hill plot of velocities corrected for Mg2+ inhibition as a function of magnesium isocitrate. In A and B the points are fitted by smooth curves and in C by the method of least squares.

1851 at the ordinate represent initial rates when all of the magnesium is converted to magnesium isocitrateand magnesium H-isocitrate. This eliminates Mechanisms i, ii, and iii except for the possibility of rapid equilibrium-ordered mechanisms with free magnesium adding first followed by free isocitrate. The rapid equilibrium-ordered mechanism gives the same initial velocity equation as a mechanism in which magnesium isocitrate is the substrate and free magnesium is a competitive inhibitor with respect to the complex. Thus, the two mechanisms cannot be distinguished by the present kinetic approach. However, since rapid equilibrium-ordered mechanisms with metal adding first occur very rarely for metal-requiring enzymes, and since metal-chelate substrates are commonly found in biological systems, the remainder of the results will be interpreted in terms of magnesium isocitrate as the active substrate for the enzyme. 2. It appears that free isocitratea-, which is the predominant form of isocitrate present under these experimental conditions, does not act as an inhibitor of the reaction, since no apparent substrate inhibition occurs at 100 mM concentrations of total isocitrate. 3. Initial velocity is a sigmoidal function of magnesium isocitrate- concentration. A replot of the ordinate intercepts of Fig. 2 as a function of reciprocal magnesium isocitrate- concentration was nonlinear, concave up, and gave a SO.~ value of 0.87 mM for magnesium isocitrate-. This experiment cannot distinguish between magnesium isocitrateand magnesium Hisocitrate as substrates, but since the experiment was done at pH 7.75, the ratio of magnesium isocitrateto magnesium Hisocitrate is approximately 1900 : 1. Therefore, if magnesium H-isocitrate is the substrate, the L&, for magnesium H-isocitrate would be 0.46 PM. The possibility that magnesium H-isocitrate is the substrate for isocitrate dehydrogenase is considered less likely than the magnesium isocitratecomplex being the substrate since the predominance of the magnesium isocitratecomplex would make magnesium isocitrate- inhibition a distinct possibility. If both magnesium isocitrateand magnesium Hisocitrate act as substrates with similar kinetic properties, only the magnesium isocitratecomplex need be considered as the magnesium H-isocitrat,e contribution to the kinetic patterns would be negligible. For the remainder of the kinetic studies described here, magnesium isocitrate- should be interpreted as being the sum of the magnesium isocitrate- and magnesium Hisocitrate complexes. Inhibition by Mg2+ in Presence and Absence of ADP-In initial experiments (Fig. 1B) lower velocities were observed for equivalent magnesium isocitrateconcentrations at elevated Mg2+ to magnesium isocitrateratios. This result suggested that free Mg+ mav act as an inhibitor of bovine heart isocitrate dehydrogenase. In order to test this hypothesis, experiments were done with magnesium isocitrateas the substrate and the concentration of free Mg*+ was varied. Evidence has been obtained in several experiments that in the absence of ADP the Hill slope for magnesium isocitrateapproaches the value of n = 2 and that plots of 1 /v against 1 /[MI12 are linear over the range of magnesium isocitrate- concentrations used in these experiments. For calculation of magnesium inhibition constants it was assumed, therefore, that the velocity of the reaction in the absence of free Mg2+ (v) can be described by = v rnSX x DfZ12 K $ [Ml]2 (11)

1 0

1.1

0.04

A [I Tl

* 0.08 ,M-

0.12

aI

FIG. 2. The effect of varying total isocitrate at fixed levels of total magnesium at pH 7.75. Concentrations of total magnesium were fixed at the levels shown by the numbers in the figure. Total nn-isocitrate [IT] varied between 10 and 100 mM. The reaction mixtures contained 0.167 M Hepes buffer at pH 7.75 and initial velocities (AA3d0 X 10 min-I) were determined as described under Methods. No activity was observed in a control experiment with 166 mM nn-isocitrate in the absence of added magnesium. The lines were fitted to the points by the method of least squares.

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were obtained. These results indicated, as has been previously shown (l), that magnesium is required for catalysis. In order to determine whether the major ionic species of isocitrate which was present in the experiment, namely magnesium isocitrateshould be tested as a potential active substrate species, the concentration of magnesium isocitratewas calculated. The relationship between initial velocity and magnesium isocitrate- concentration is shown in Fig. 1B. Since there appears to be a correlation between reaction rate and magnesium isocitrateconcentration (Fig. 1B) as well as between rate and free isocitrate concentration, the following species were considered as possible substrate requirements for the isocitrate dehydrogenase reaction: (i) free Mg2+ and free isocitratea-; (ii) free Mg*+ and free H-isocitrate2-; (iii) free Mgti and magnesium isocitrate-; (iv) only magnesium isocitrate- complex; (v) only magnesium H-isocitrate complex; and (vi) both magnesium H-isocitrate and magnesium isocitrate- can be substrates, The results of Fig. 1 do not eliminate any of the above possibilities, but suggest that there is an interdependent mechanism of substrate addition since plots of initial velocity against either free isocitrate or magnesium isocitrate- appeared sigmoidal. Requirement of Free JZagnesium as Substrate-It has recently been reported that H-isocitratezand free Mgfi are the required substrates for the pig heart isocitrate dehydrogenase (9). In order to test the hypothesis that free magnesium is required for the bovine heart enzyme, initial velocities were measured at pH 7.75, where the ratio of isocitrate+ to H-isocitrate* would be expected to be 100. In this experiment, illustrated in Fig. 2, the concentration of total isocitrate was increased at fixed magnesium concentrations so as to increase the concentration of magnesium isocitrate- while decreasing the concentration of free magnesium. It will be apparent that at the ordinate intercepts in Fig. 2, the free Mg2+ concentration approaches zero. Since the ordinate intercept values are strongly dependent on the concentration of total magnesium the following conclusions can be reached. 1. Any reaction mechanism which requires finite concentrations of free Mg2f can be eliminated since the observed velocit,ies

Double reciprocal plots of initial velocity as a function of [MZ12 at pH 6.65 and 7.2 yielded intersecting lines (Fig. 3) which gave

1852
good fits to the equation for competitive inhibition: (12) where K, is the apparent Michaelis constant of magnesium isocikate and f(i is the inhibition constant of free Mg2+. As indicat,ed in Equations 13 and 14, the methods for calculat,ion of inhibition correction factors differ in the absence or presence of ADP, and at pH 7.2 the numerical values of the con&&s obtained and the units of K are not the same (Table I). Nevertheless, at comparable concentrations of Mg2f and magnesium isocitrate the inhibition correction factors are similar, when calculated from the constants of Table I. This implies, at least at pH 7.2, that the relative kinetic constants for Mg2+ and magnesium isocitrate are not changed substantially by the modifier. Initial Velocity as Function of Magnesium Isocifrafe- Concentration-when the correction factors from Table I are applied to experiments where several fixed levels of magnesium isocitrate had been maint.ained by varying concentrations of free isocitrate and free Mg*+ at pH 7.2 (Fig. lB), the corrected velocities are dependent only on magnesium isocitrateconcentration. The data of Fig. lB, corrected for Mg2+ inhibition, are shown as a Hill plot in Fig. 1C. A Hill coefficient of 1.6 and an SO.s value of 0.75 mM was obtained from the data. The results of similar experiments at pH values of 6.65, 7.2, 7.4, and 7.75 are summarized in Table II. The X0.s for magnesium isocitrateincreases from 0.18 to 0.95 mM as a function of pH, while the Hill coefficient remains constant near 2.0. Experiments at pH 7.2 using u-isocitrate as substrate gave approximately the same Hill coefficient as when nn-isocitrate was used (1.8 and 2.0, respectively). The LS&, value for Disocitrate was one-half of that found when nn-isocitrate was used as substrate. This result is consistent with the previous observation that n-three-isocitrate is the substrate for DPNdependent isocitrate dehydrogenase (1). Activating Species of ADP-In the above studies, evidence has been presented that magnesium isocitrate is the substrate for the enzyme. Since ADP is a relatively strong chelator of magnesium, it became of interest to know whether the free or the chelated forms of ADP are active modifiers. An experiment similar to that shown in Fig. 2 was designed in which free hilg2+ and magnesium ADP complex are effectively removed by adding free isocitrate in large excess. The effect of increasing concenkations of isocitrate at constant total magnesium (0.10 mM) was studied at several fixed levels of ADP, and plotted as 1 /v against reciprocals of isocitrate concentration (Fig. 4A). As free isocikate increases, the magnesium is converted to magnesium isocitrate and velocity increases. For example, without ADP, the concentration of magnesium isocitrate rises from 0.087 to 0.096 mM as nn-isocitrate increases from 12.5 to 50 mM. At 50 mM isocitrate with varying ADP, the concentration of free h/Ig2+ is reduced to between 3.4 and 3.7 PM and only about 1% of total AD1 is present as MgADP(2 to 8 PM). Extrapolation to the ordinate leads to di$erent intercept values, indicating that free ADl can activate isocitrate dehydrogenase. If MgADPwere the active species all lines would intersect at a common point on the ordinate. Furthermore, as there is no tendency for the lines to curve upward as isocitrate concentration increases, free isocitrate does not act as an inhibitor with respect to magnesium isocitrate. If such inhibition did occur it should be observed in this experiment (as well as in Fig. 2) since the ratio of free isocitrate to magnesium isocitrate is in excess of 500. This experiment also confirms the previous results which indicated that free magnesium is not required for the reaction, The inhibition correction factors brackets in Equations 13 and 14. are the terms shown in

where &I is the magnesium isocitrate complex, JZ is free Mg2+, Kmic is the magnesium inhibition constant, and K is an apparent constant which should be equivalent to (X0.5)2 of magnesium Correction for inhibition by free Mg2+ can then be isocitrate. expressed by the relationship

The kinetic con&ants obtained from the computer analyses are recorded in Table I. These constants could be used in Equation 13 to calculate the uninhibited initial velocities of reactions containing inhibitory levels of free Mg2+. Between pH 7.2 and 7.75 in the presence of relatively high concentrations of ADP the Hill coefficients for magnesium isocitrate- approach values of n = 1. In an experiment similar to that described in Fig. 3 inhibition by Mg2f at pH 7.2 was tested in the presence of 0.3 mM ADP and was found to be competitive with magnesium isocitrate. The constant,s obtained from fits of the dat,a to Equat.ion 8 are reported in Table I. In the presence of the modifier the correction of velocity can be expressed in the usual way by

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(14)

12

16

FIQ. 3. Inhibition by Mg*+. A and B, double reciprocal plots of velocity versus [magnesium isocitrate]* (MI). The numbers above the lines refer to concentrations of free Mg2+ as millimolar. The lines were fitted to the points by a computer program (19) and Equation 8. A, pH 7.2; B, pH 6.65. The constants derived from these fits are reported in Table I,

1853
TABLE I from ncagnesium inhibition are given in Fig. 3. Calculations K i
he

Constants

Examples
ADP

of the conditions

of incubation

experiments are described

Vmx zt S.E.

in the text and Equations

13 and 14.
range

K,,,ic f SE.
?nM

SE.

[Magnesium

isocitrate-] range

[Free Mgs+l

.Aaro mine mu
?nM

?nM

0 0 0.3

6.65 7.2 7.2

0.0210 zt 0.0091 1.83 f 0.20b 0.79 f 0.10

0.0189 f 0.239 f
rnM

0.0087 0.041* 0.006

0.0427 f 0.004 0.096 * 0.004* 0.105 III 0.003

0.15~0.30 0.25-1.0 0.065-O. 39

0.0250-0.15 0.026-4.0 0.04-l .5

0.093 f

@Values expressed in terms of concentrations of magnesium DL-isocitrate. In experiments where the D isomer is used the value of 4X = Sa.s as dethe constant is one-fourth and one-half that reported here in the absence and presence of ADP, respectively. scribed by Equation 11. b Weighted mean from three experiments. since extrapolation to t.hc ordinat,e gives t.he velocity at zero free magnesium. These intercept values represent the initial velocity at 0.1 mM magnesium isocitrate, zero fret magnesium, zero MgADP-, and the free ADP3- concentrations shown in Fig. 4A. The lines in Fig. 44 show approximate linearity over the experimental range of magnesium isocitrate employed, since its concentration changed only by small increments (from 0.067 to 0.096 mM), however, over wider concentration ranges such plots would not be linear. A secondary plot of the reciprocals of the differences of the velocities at the intercepts (l/v - vO) against the reciprocals of concentrat.ions of ADP yields a straight line which intersects the abscissa at -l/K,,, (Fig. 4B). The data were fitted to Equation 10 as described under Methods and an apparent K, of 0.63 mM has been calculated for free ADP. In another experiment, where the concentrations of ADP were lower (0.15 to 0.20 mM), the value of K, was 0.68 mM. The results above suggested that free ADP can activate the enzyme; however, they leave uncertain the activity of t,he magnesium ADP complex. The react,ion was studied in an experiment where magnesium m-isocitrate was maintained at the same concentration as that at the intercepts of Fig. 4A (0.10 mM), free Mg*+ was kept constant and total ADP was varied between 0 and 2 mM. When free Mgz+ was constant at 0.217 or 0.434 mM the ratio of [free ADP] to [magnesium ADP] was fixed at 2 or 1, respectively. Comparison wit,h K, values for free ADP obtained under the experimental conditions of Fig. 4 should give an indication of the activity of magnesium ADP. Thus, similar const.ants would indicate that magnesium ADP is inert; if K, of free ADP is lower or higher than that of Fig. 4 magnesium ADP would be an activator or an inhibitor, respectively. The results of such an experiment for total ADP arc represented in Fig. 5A in the form of a rectangular hyperbola with a positive ordinate intercept. The experimental points were fitted to Equation 9 to give values of ~0, KN, and KD; the latter represents the apparent K, for ADP. The values of apparent K, of total ADP are 0.94 and 1.14 mM where ratios of [free ADP] to [magnesium ADP] are 2 and 1, respectively. However, the constants of free ADl are essentially the same (0.63 rnhf versus 0.57 mM) (Fig. 5B), and comparable to the value of K, (0.63 mM) obtained under the experimental conditions shown in Fig. 4. These results suggest that free ADP is the active modifier and that magnesium ADP is inert. The results from Figs. 4 and 5 and of additional experiments are summarized in Table III. Relationship between Substrate and ADP--In studies similar to those shown above, the values of K, of free ADP were found to vary considerably depending on the concentration of substrat,e. Thus, at pH 7.2 the values of K,, of free ADP were 0.6 and 0.2 mM with 0.10 and 0.2Gl mM magnesium nr,-isocitrate, respectively. It became of interest, therefore, to study the interdependence of substrate and modifier. In these experiments free Xg2+ was held constant at relatively low and uninhibit,ory levels (0.025 to 0.05 mnf); magnesium isocitrate and free ADP were varied by appropriate changes in isocitrate and total ADP. The results of experiments at pH values of 6.65, 7.2, 7.4, and 7.75 are presented in Table II. The experiments at pH 7.2 and 7.4 show the apparent K, of ADP decreasing with increasing concentrations of magnesium isocitrate, and So.5 of magnesium nL-isoeitrate declining wit.h increasing AD1 (Table II). The value of the Hill slope declines progressively from n = 1.9 in t.he absence of ADP toward n = 1.0 as t.he concentration of ADP increases. The same trend in values of slopes is noted in experiments at pH 7.75. At pH 6.65 t.he slopes of the Hill plots for magnesium isocitrate are near n = 2 and unaffected by ADP in the concentrat.ion range 0 t.o 0.2 mM. This has been confirmed by the parabolic shape of plots of l/v versus l/[magnesium isocitrate-] with the fixed levels of ADP shown in Table II. It is possible, however, that higher concentrations of ADP are necessary at pH 6.65 in order to decrease the slope of Hill plots to 1.0. It may be that there is no interdependence between substrate and ADP, as suggested by the lack of response of the Hill slope (n), however, in the absence of data on inhibition by Mg2f in the presence of ADP, and in view of the fact that, Mg2+ appears to inhibit more strongly at low pII values, no firm conclusions can be reached. 13~ the same criteria, it is difficult to determine whet.her or not the kinetic constar1t.s of ADP and substrate are completely independent at pH 6.65 (Ta.ble II), The effect of magnesium isocitrate concentrat,ion on ,!!& of ADP has also been examined in Hill plots. Slopes (m) with values near unity have been obtained at. pH 6.65, 7.2, 7.4, and 7.75 (Table II). The values of m are not affected by changes in magnesium isocitrate concentration. Eject of pa-Increasing pH between pH 7.2 and 7.75 leads to relatively small increases of K, for magnesium isocitrate and ADP; inhibition by free Mg2+ becomes less severe and V,, is either the same or shows a small increase (Tables I and II). The constants at pH 6.65 are markedly different from those between pH 7.2 and 7.75, showing in the absence of ADP much lower So.5 for magnesium isocit,rate, lower Vmsx, and much more severe inhibition by free Mgz+. The inhibition by Mg2+ as a function of pH is of special interest in view of the proposal of Cohen and Colman that in-

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1854
TABLE

II
110 100 so I v 7 .E E g 7 x 60 70 60 50

hflect of varying ADP on So.5 of magnesium isocitrate and of changing magnesium isocilrate complex concentrations on K,,, of ADP At pH 7.2, 7.4, and 7.75 the incubation mixtures contained 0.167 M sodium Hepes buffer, free Mgz+ was held constant at 0.05 M, and the concentrations of magnesium isocitrate were maintained at the levels indicated by appropriate changes in DLisocitrate. At pH 6.65 the incubation mixtures contained 0.167 M Pipes buffer and 0.025 M free Mg*+. Concentrations of ADP and values of K,,, of ADP are reported as total ADP. Concentrations of magnesium isocitrate (MI) and values of So.5 are on the basis of nn-isocitrate. Values of K,,, of ADP are calculated from the computer fits of the data to Equation 9 as described in the text. The slopes (m) were determined in Hill plots. Values of SO.5 of magnesium nn-isocitrate and of slopes (n) were calculated from Hill plots at pH 7.2 and 7.4. At pH 6.65 an average value of V msx of 0.0314A Aa40 min-1 was used for calculations of the Hill plots; it was obtained from plots of l/v versus l/[magnesium isocitrate-je at various fixed levels of ADP (range of V,,,,, was from 0.0337 to 0.0305 A As& min-I). Incubation conditions and the calculation of concentrations of components of the reaction mixtures from dissociation constants of magnesium isocitrate and of magnesium ADP were as described under Methods.
-7 pH 6.69

ADP,mM 0

30 20

A
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0 0.D2 0.04 VDL-ISOCITRATE

pH 1.2 nz icope 1.0 1.0 1.2

0.27 0.22 0.16 0.13

pH 1.4

PI3 1.15 m ?n slope

t--vime
FIG.

(TOTAL),mM-

WI

-n&M

A%P 7m.u

A%

m slope

A% e&M

i%P

SlC+C

0.10 0.15
0.20 0.30 0.40 0.50 0.60 0.75 0.80 1.50

0.16 0.23 0.21

1.0 1.0 1.2 1.0 1.2

0.44
0.34 0.29 0.22 0.16

1.1 0.9 1.0 0.26 1.0 0.22 1.1 0.49

1.1 1.1 1.0

n

4. Response of velocity at constant total magnesium and varying isocitrate at several fixed levels of ADP (pH 7.2). The concentration of total magnesium was 0.10 mM, total nn-isocitrate was varied from 12.5 to 50 mM, and ADP concentrations were 0, 0.20, 0.25, 0.33, 0.50, and 1.00 mM. A, double reciprocal plot of velocity versus total nn-isocitrate. B, secondary plot of A in which the differences between the velocities at the intercept in the absence (V,) and presence of ADP (V) are plotted as the reciprocals l/(V - V,) versus l/ADP. The intercept at the base-line is equivalent to -1/K, of free ADP. The lines to which the experimental points were fitted were constructed by the computer program for a rectangular hyperbola (19) and Equation 7. hibition of pig heart isocitrate dehydrogenase occurs at lower values of pH due to an increase in the concentration of the manganous complex of dibasic isocitrate (9). If this were the case for the magnesium isocitrate complex in the present studies, the apparent inhibition constant for magnesium would be expected to decrease approximately 4-fold as the pH decreases from 7.2 to 6.65, In fact, the magnesium inhibition constant decreases by a factor of 85 over this pH range (Table I) which makes the simple explanation of magnesium H-isocitrate formaAt present the reason for such a tion and inhibition unlikely. large change in the inhibition constant is unknown, but it could be due to a pH-dependent conformational transition in the protein over such a pH range.
DISCUSSION

--s0.1
md(

[ADPI 7md 0
0.05 0.10 0.15 0.20 0.30 0.50 1.00

Ml
1

n
ilope

0.6 Ml
nzM

)t

0.6 Ml ?nM

n
SlO$%

So.s MI
mld

slope

&PC
2b

0.18 0.12 0.11

0.10 0.10

2.0 2.0 2.0

2.0

0.41
0.30 0.25

1.9 1.7 1.6 1.6 1.5 1.4 1.3

0.54 0.46 0.38 0.29 0.23 0.17

2.0 1.9 1.7 1.6 1.5 1.4 1.3

0.956

1.9

-[ADPI WZM 0 cd

0.19 0.16 0.12 0.09

0.10 V,,X
AAsro

0.63c 0.77c 0.87~

1C 1C 10

-AAsao min-

V rnP.x
AApo v&r-

V,.X
AAaro min-

mitt-l

--

0.0305 ().044-0.051

0.115 0.0849

0.0817 0.0887

0.115 0.13-0.2~

(LResults uncorrected for inhibition by 0.025 mM Mg2+. The velocity correction factors for magnesium inhibition at 0.1, 0.2, and 0.3 mM of magnesium isocitrate- are 1.20, 1.37, 1.76, respectively. * From plot l/v versus l/[magnesium isocitrate-12. 0 From plot l/v versus l/[magnesium isocitrate-1. At 0.3, 0.6, and 1.0 mM ADP the values of V,,, were 0.15, 0.18, and 0.2, respectively. d Calculated from Equation 9 except where indicated.

Steady state kinetic studies on the role of magnesium in the mechanism of bovine heart DPN-linked isocit.rate dehydrogenase are consistent either with magnesium isocitrate being the actual substrate or with a rapid equilibrium-ordered mechanism with free magnesium adding before the addition of free isocitrate. Since mechanisms of the latter type are very uncommon for metal-requiring enzymes, the former mechanism was assumed for the purpose of discussing the results and postulating a reaction sequence for the substrate and modifier of bovine heart isocitrate dehydrogenase. The proposed substrate function of magnesium isocitrate is in accord with the earlier observation that a combination of divalent metal ion activator and n-iso-

1855
, I

V,=O.O69

l, ,

I K-*1.14 111 .

1.0

1.5

~/FREE AknM-1

2.6

TOTAL AOP,mM

FIG. 5. Activity as a function of ADP at fixed ratios of free ADP to magnesium ADP at pH 7.2. Magnesium nn-isocitrate was constant at 0.10 mM. Total ADP was varied and the ratios of [free ADP] to [magnesium ADP] were fixed at 1 or 2 by maintaining free Mg*+ at 0.434 mM (Curve 1) and 0.217 mM (Curve B), respectively. A, velocity as a function of total ADP. The experimental points were fitted by a computer program (19) to Equation 9. B, double reciprocal plot of (V - VO) versus free ADP, where V, is the velocity in the absence of ADP. The points were fitted by the method of least squares. The intercept at the base-line is --1/K,,,. At 0.217 and 0.434 mM Mg2+ the inhibition factors? are about 1.1 and 1.3, respectively. The inhibition of velocity is reflected in the differences in calculated I,,, for Curves 1 and 2 shown in Panel A and the differing slopes and ordinate intercepts (with practically identical base-line intercepts) of the lines in Panel B. TABLE

III
by total ADP and free ADP

Comparison

of activation

concentration was examined in most earlier studies of the kinetics of this enzyme (2, 4, 6, 8). The present results suggest that such determinations of apparent kinetic constants, depending substantially on lower concentrations of isocit.rat.e, tend to be imprecise because of substantial apparent inhibition by free Mg2+ at the relatively high [Mg*+] to [magnesium isocitrate-] ratios. Since reported values of Hill coefficients are particularly variable, experiments (not shown here) have been done at pH 7.2, 7.4, and 7.6 in which total magnesium was fixed at 6.6 mM and total nn-isocitrate was varied from 0.4 to 40 mM. Hill plots of the results as a function of magnesium isocitrate concentrations gave Hill coefficients (n) ranging from 1.9 to 2.0; the values in the absence of ADP are thus in agreement with those report.ed in Table II. However, plots of the same experimental data as a function of total isocitrate yielded Hill coefficients considerably lower than n = 2 (1.3 t.o 1.4). These differences in part reflect the fact that with constant total magnesium (6.6 mM) and changing isocitrate (total or free) the ratio of [isocitrate]/[ma.gnesium isocitrate] is not constant (Equations 2 and 3) but rises with increasing isocitrate concentration. The elucidation of active substrate and modifier forms in t.he present study eliminates a number of mechanisms which had previously been considered. Among t.hese are separate combinations of free Jlg2+ and free isocitrate at the substrate site, except for the rapid equilibrium ordered possibi1it.y previously discussed, and t,he combination of the Mg;ADPcomplex at the modifier site. Although the experiments presented in this paper were not designed for quantitative analysis of the reaction mechanism, the results do permit the formulation of a reaction mechanism which shows qualitative agreement with the experimental results. Such a mechanism is presented in Scheme I, where E represents enzyme saturated with DPNf.
MI\ E\ MI, E*MI Eu (MI),

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Magnesium nn-isocitrate (0.10 mru) was constant at pH 7.2 in all experiments. Free Mg2+ and ADP varied as indicated. The data were obtained in experiments as described in Figs. - 4 and 5.
Y-

w
ADP \I1

------A

\r
PRODUCTS

Free MP
~___ ?nM

Free ADP to MgADP-

Total ADP range

?nM

Km of total mu f

ADP S.E.

K,,, of Free ADP

vim f S.E.

0.1 0.217 0.434

47034 2 1

0.2-1.0 0.3-1.5 0.2-1.0 0.4-2.0

0.63 0.84 0.94 1.14

f f f f

0.06 0.07 0.07 0.06

0.63 0.68 0.63 0.57

f f f f

0.06 0.05 0.05 0.03

The over-all initial velocity written in general form as:

equation

for Scheme I can be

citrate is required for protection against inhibition by low concentrations of sulfhydryl group-binding agent.s (2, 13) while either the metal ions or isocitrate alone were ineffective. It was reported previously that manganese is an inhibitor of the enzyme (8, 9), but such an effect was not observed with magnesium (8). The present studies are consistent with the idea that inhibition by free Mg2+ can occur; that it is competitive with magnesium isocitrate-, and that the inhibition becomes more severe with decreasing pH (Fig. 3 and Table I). In contrast to the requirement for the combination of magnesium and isocitrate as substrate, only the free form of the modifier is active (Figs. 4 and 5; Table III). Previous kinetic investigations of mammalian DPN-specific isocitrate dehydrogenase were handicapped by lack of information on the nature of the ionic species which interact with the enzyme, and interpretation of kinetic results became somewhat ambiguous. Thus, the effect of total isocitrate concentration on velocity with constant relatively high divalent metal ion

V(IMZP + bL4DPINfII)
= [MZ]2 + c[MZ] + d[ADPJ + e]ADPI]MZl + j

(15)

where b, c, . . f represent kinetic constants. In the absence of the modifier, ADP3-, only the upper reaction sequence of Scheme I would operate, and Equation 15 would reduce to:
V[MZ]2 v = Mifz12 + c[MZl + j

(16)

This ordered combination of 2 substrate molecules is consistent with the results of initial velocity studies, which gave good fits (19) to Equation 16,* i.e. parabolic plots of l/v against l/magnesium isocitrate- and Hill plots for magnesium isocitrate- with slopes near 2 in the absence of ADP. These kinetic features may be most simply explained by the existence of two interdependent sites for substrate combination, both of which must be occupied before products can be released. It is thought that * T. Aogaichi, V. L. Sehramm, and G. W. E. Plaut (1972) unpublished observations.

1856 initial binding of substrate induces a change in the enzyme which transforms the remaining site (or sites) into a catalytically active conformation. In the absence of substrate these sites are presumed to be identical since previous observations indicate that the enzyme is composed of eight identical subunits (15). Since steady state kinetic data cannot be used to determine the stoichiometry of substrate binding, additional experiments will be required to determine the actual number of each type of binding site on the enzyme. In the absence of such quantitative data no meaningful consideration can be given to binding at multiple identical sites. Such combinations would not change the general form of Equation 16, but would have the effect of changing the kinetic constants by a statistical factor for multiple interactions. In contrast to the interdependence of enzyme-substrate interactions in the absence of ADI-, kinetic studies in the presence of relatively high concentrations of the modifier are consistent with a single (or multiple independent) combination of substrate with enzyme since the data give good fits to the equation describing a single such combination (Equation 7). Such results are consistent with the pattern predicted from Equation 15, which reduces to the same form as Equation 7 as ADP concentration becomes very high. This interaction is shown in Scheme I, and leads to the formation of the ADP .enzyme .magnesium isocitratecomplex. Saturation of the modifier site (or sites) causes the enzyme to exhibit independence of substrate sites, with an apparent loss of the requirements for the catalytically inactive site (or sites) to be filled before catalysis can occur. This phenomenon could be explained by ADP causing such an appreciable increase in affinity between substrate and the noncatalytic site (or sites) that saturation occurs at all experimental concentrations of magnesium isocitrate. Alternatively, in the presence of ADP, binding of substrate at the noncatalytic site may no longer be required for catalysis. The latter postulate constitutes the simplest mechanism and has been illustrated in the sequence of reactions from E to the formation of ADI.E. magnesium isocitrate-. A third possibility is that ADP causes both sets of sites to become catalytically active, equivalent, and independent. The latter seems less likely t,han the previous postulates since the formation of additional cat.alytic sites might be expected to lead to increased activity. However, the results indicate that Fmnlr is independent of modifier concentration (Tables I and II). Indications for binding of free ADPa+ to the free enzyme have been obtained in gel filtration esperiments (3) and by the demonstration that the modifier causes protein concentrationdependent dimerization of the enzyme (15) in the absence of added divalent metal ions and substrates. The kinetic data presented here are consistent with these earlier results, and also show that modifier combines in an independent manner at its respective site (or sites), as the experimental data fit well to It Equat.ion 17, which describes a single combination of ADP. will be noted that Equation 17 has t.he same general form as Equation 9. r I + e[ADPl 1 Cd + eM4ZIL4I~Pl (17) l + lMZIZ f c[MZl + f This prediction is confirmed by the data (Table II). Although Scheme I is capable of explaining the kinetic properties of DPN-linked isocitrate dehydrogenase from heart, it should be emphasized that this is the simplest mechanism consistent with the data and in no way precludes more complex reaction pathways which may well become apparent with further kinetic and thermodynamic studies. Indeed, isocitrate dehydrogenases from other sources appear to exhibit more complex mechanisms than that proposed here (11, 20, 21), although failure to identify the active substrate form could have added an additional degree of complexity to the kinetic results. The effects of a number of factors which may regulate mitochondrial isocitrate oxidation at the stage of DPN-linked isocitrate dehydrogenase have been discussed previously (for review see Ref. 13). The present results emphasize the need to consider the level of intramitochondrial divalent metal ion (or ions) (e.g. Ref. 22) on the flux of metabolites at this step, since free Mg*f or M& inhibits the reaction and the rate of reaction is dependent on the concentrations of the active species of substrate (e.g. magnesium isocitrate) and modifier (free ADP3-). In addition, fluctuations in intramitochondrial pH, which may occur in various metabolic states (23, 24), could affect the rate of catalysis since some of the kinetic constants are particularly sensitive to changes in pH (Tables I and II). REFERENCES
1. PLAUT, G. W. E., AND SUNG, S-C. (1954) J. Biol. Chem. 207, 305-314 2. CHICN, R. F., AND PLAUT, G. W. E. (1963) Biochemistry 2, 1023-1032 3. CHEN, R. F., BROWN, D. M., AND PLAUT, G. W. E. (1964) Biochemistry 3, 552-559 4. GOEUILL, II., AND KLINGENBERG, M. (1964) Biochem. 2. 340, 441-464 5. KLINQENBERG, M., GOXBELL, H., AND WENSKE, G. (1965) Biochem. 2. 341, 199-223 6. STUN, A. M., KIRKMAN, S. K., AND STEIN, J. H. (1967) Biochemistry 6, 3197-3203 7. STEXN, A. M., STEIN, J. H., AND KIRICMAN, S. K. (1967) Biochemistry 6, 1370-1379 8. PLAUT, G. W. E., AND AOGNCHI, T. (1968) J. Biol. Chem. 243, 5572-5583 9. COHEN, P. F., AND COLMAN, R. F. (1972) Biochemistry 11, 1501-1508 10. HARVEY, R. A., HERON, J. I., AND PLAUT, G. W. E. (1972) J. Biol. Chem. 247, 1801-1808 11. ATKINSON, D. E., H.~THAW.LY, J. A., AND SMITH, E. C. (1965) J. Biol. Chem. 240, 2682-2690 12. SANWALL, B. D., AND COOK, II. (1966) Biochemistry 6, 886-894 13. PLAUT, G. W. E. (1970) Curr. Top. Cell. Regul. 2, l-25 14. PLAUT, G. W. E. (1969) Methods Enzymol. 13, 34-42 15. GIORGIO, N. A., JR., YIP, A. T., FLF:MING, J., AND PLAUT, G. W. E. (1970) J. Biol. Chem. 246, 5469-5477 16. SIEGEL, J. M., MONTGOMERY, G. A., AND Bocq R. M. (1959) Arch. Biochem. Biophys. 82, 288-299 17. GHZYUOWSKI, A. K., TATI,:, S. S., AND DATTA, S. P. (1970) J. Chem. Sot., Sect. A, 241-245 18. OSULLIVAN, W. S., AND PNUUN, D. L). (1964) Biochemistry 3, 18-26 19. CLELAND, W. W. (1963) Nature 198,463-465 20. ATKINSON, 11. E. (19G6) Annu. Rev. Biochem. 36, 85-124 21. S.~N~.UL, B. D., STXHOW, C. S., AND COOK, It. A. (1965) Biochemistry 4, 410-421 K., AND WOJTCZX<, L. (1971) Biochem. Biophys. 22. BOGUCKA, Res. Commun. 44, 1330-1337 B. (1967) in Biochemistry of Mitochondria (SLATER, 23. CHANCF:, E. C., KANIUGA, Z., AND WOJTCZAK, L., eds) p. 93, Academic Press, New York 24. ADDANKI, S., CAHILL, F. D., AND SOTOS, J. F. (1968) J. Biol. Chem. 243, 2337-2348

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21 = V 1

Furthermore, Equation 17 predicts that the apparent K, values for ADP- will be dependent up011 the concentration of substrate present, as
K, for ADP =

[MZI~ + c[MIl + f d + elMI