THEJOURNAL OF BIOLOOICAL CHEMISTRY 0 1988by The American Society for Biochemistry and Molecular Biology, Inc

Vol. 263, No. 35, Issue of December 15. pp. 186411-18649,1988 Printed in U.S.A.

The Catalytic Site of Rat Hepatic Lauric Acid o-Hydroxylase

PROTEIN VERSUS PROSTHETICHEME FATTY ACIDS* ALKYLATION IN THE w-HYDROXYLATIO~ OF ACETY~ENIC

(Received for publication, May 16, 1988)

Claire A. CaJacobS, William K.ChanS, Elizabeth Shephardg, and Paul Ortiz de o n t e l l ~ o S ~ R. M

From the ~ ~ e p a r tof ~n ~ r ~ e u t i c a ~ School of Pharmacy, and Liver Center, University of California, ~ t Chemistry, Sun Francisco, California94143 and the WDemrtment of ~ ~ ~ ~ r n U n i tv e r,College, h n d o n WC1E 6BT, i s ~ ~~~ United Kingdom

Cytochrome P - 4 5 0 ~ ~ purified from clofibrate-in- but it is clear from the available evidence that distinct isoduced rat liver oxidizes lauric acid to 11- and 12- zymes catalyze the different w-hydroxylations.The lauric acid a hydroxydodecanoic acid in approximate~y1:17 ratio w-hydroxylases from rat liver (2) and pig kidney (3), and the at a rate of 2 0 nmolJnmol P-450Jmin. In contrast, prostaglandin w-hydroxylases from rabbit liver ( 5 ) , lung (6, cytochrome P-450b oxidizes lauric acid much more 71, kidney (8),and intestine(9) have been extensively purified, slowly (0.5 nmol/nmol P-4FiO/min) to an 8 l mixture albeit not in all cases to homogeneity. The lauric acid w : of the same metabolites, Western blot analysis indi- hydroxylase preparation of Tamburini et al. (2) was recently cates that P - 4 5 0 ~ ~ ~ accounts for 1-2 and 16-30%, reported by Hardwick et al. (15) to contain two proteins. The respectively, of the total cytochrome P-450 in uninduced and clofibrate-induced rat liver. Cytochrome b6 latter investigators have isolated a cDNA clonethat codes for increases theefficiency of w-hydroxylation but not the one of the two proteins, have sequenced it, and have demonrate of catalytic turnover. Incubation of the enzyme strated that iti s a lauric acid w-hydroxylase by expressing it with 10-undecynoic acid (10-UDYA) results in loss of in catalytically active form in yeast. Quantitation of the approximately 45% the enzymatic activity butnone hepatic lauric acid w-hydroxylase by radial immunodiffusion of of the enzyme chromophore. Approximately 1 mol of led Bains et al. (14) to suggest that 22 and 57% of the total 1,II-undecandioic acid is produced per mole of inac- cytochrome P-450 in the liver, respectively, of uninduced and tivated enzyme. This extraordinary inactivation effi- clofibrate-induced rats is the lauric acid w-hydroxylase. The fatty acid w-hydroxylases are particularly interesting ciency is confirmed by NADPH consumption studies. Aproximately 0.5 equivalents of label are covalently from a mechanistic point ofview because they oxidize the bound to the enzyme when it is incubated with *C- thermodynamically disfavored terminal methyl group. Model labeled IO-UDYA. 11-Dodecenoic acid appears not to studies with hypervalent oxometalloporphyrins show that oxbe a substrate for cytochrome P-450LAa but is oxidized, idation of the hydrocarbon methylenes is highly favored over presumably by a contaminating isozyme, to a 1O:l oxidation of the terminalmethyl group (16-19). This inherent mixture of 11,12-epoxydodecanoic acid and 12-oxo- reactivity difference results in preferential w-1-hydroxylation dodecanoic acid. The results suggest the presence of of hydrocarbon chains by relatively nonspecific cytochrome two closely related P - 4 5 0 ~ ~ enzymes, only one of P-450 isozymes ( e g . rat P-45&, rabbit P - 4 5 0 ~ ~ 2 ) (20-23). which is susceptible to inactivation by IO-UDYA. They Preferential hydroxylation of the methylene groups is prealso indicate that cytochrome P - 4 5 0 has ~ highly dicted by the now favored hydrogen radical abstraction mech~~ a structured active site that sterically suppresses W-1-anism (reviewed in Ref. 24) if one considers that the C--H hydroxylation in order to deliver the oxygen to the bond strength decreases in the order primary > secondary > thermodynamically disfavored terminal carbon. Pro- tertiary. The bond dissociation energies for removal of a tein rather than heme alkylation follows from this hydrogen to give the methyl, isopropyl, and t-butyl radicals reaction regiospecificity. are thus, respectively, 98.0, 94.5, and 91.0 kcallmol (25). wHydroxylases must therefore override the inherentspecificity of the catalytic species for the weaker C-H bond. Hepatic cytochrome P-450 isozymes oxidize terminal acetA number of the known isozymes of cytochrome P-450 are designed to specifically w-hydroxylate long or medium length ylenes (RC=CH) to ketenes (RCH==C=O)that add water to fatty acids (1-4) as well as the structurally related prostaglan- give acetic acid derivatives (RCH2C02H) (26). The cytodins (1, 5-9), leukotrienes (10, ll),prostacyclins ( E ) , and chrome P-450 isozymes involved in the reaction are simultathromboxanes (13). The number, detailed specificities, and neously inactivated (27, 28). In most instances, inactivation ~ h y s ~ o l oroles of these w-hydroxylases remain ambiguous, is due to N-alkylation of the prosthetic heme group of the ~ca~ enzyme by the catalytically activated acetylene. The heme N* Support for this research was provided by National Institutes of alkyl group obtained in the oxidation of R - C e H i s invarHealth Grant GM 25515, the Cancer Research Campaign and the iably NCH2COR. The possibility that acetylenes are also Medical Research Council of GreatBritain, and NorthAtlantic oxidized to species that react with the protein matrix is Treaty Organization Travel Grant 86/0789. The core facilities of the University of California Liver Centerare supported by National suggested by the disparity between the decrease in the specInstitutes of Health Grant P-30 AM 26743. The costs of publication The abbreviations used are: heme, iron protoporphyrin reof this article were defrayed in part hy the payment of page charges. in This article must therefore be hereby marked ~ V e r ~ i s e ~ e R t gardless of the iron oxidation and ligation states; SDS-PAGE, polyacrylamide gel electrophoresis in the presence of sodium dodecyl accordance with 18 U.S.C. Section 1734 solelyto indicate this fact. sulfate; DETAPAC, diethylenetriaminepentaaceticacid; 10-UDYA, 1[ To whom correspondence should be addressed: School of Pharmacy, S-926, University of California, San Francisco, CA 94143-0446. 10-undecynoicacid; HPLC, high pressure liquid chromatography.

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18640

Lauric Acid o-HydroxyZase Protein Versus Heme A ~ k ~ l u ~ i o ~

troscopically measured content of cytochrome P-450 and the loss of aryl hydrocarbon hydroxylase activity observed by Gan et al. (29,30) in i n ~ b a t i o n of liver microsomes with ethinyls substituted polycyclic aromatic hydrocarbons. We have demonstrated that insertion of a terminal triple bond into the hydrocarbon chain produces fatty acid analogues that are highly selective mechanism-based inactivators of the fatty acid w-hydroxylases (31, 32).Inactivation of the rat liver lauric acid w-hydroxylases bythese agents has been found to result in negligibleloss of the cytochrome P-450 chromophore. This can be explained either by the presence of a very low titer of the w-hydroxylases or by inactivation via a mechanism that does not cause chromophore destruction. TO clarify this question and to investigate the mechanism by which the enzyme promotes the intrinsically more difficult ohydroxylation, we have purified lauric acid o-hydroxylase, quantitated it by Western blotting procedures, identified the products formed on oxidation of terminally unsaturated fatty acids, and investigated the mechanism of its inactivation by a radiolabeled acetylenic inhibitor. A part of this work has been reported in preliminary form (33, 34).
MATERIALS AND METHODS* RESULTS

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tlme (mln)

FIG. 5. Inactivation of purified, reconstituted cytochrome P-450Lh by preincubation with 10-UDYA in the presence and absence of NADPH. The loss of lauric acid a-hydroxylase activity observed by preincubationwith 10-UDYAin the presence of NADPH has not been corrected for loss observed in the absence the of NADPH. The experimental details are given in Materials and Methods (Miniprint).Little loss of activity is observed if the enzyme is preincubated without10-UDYA.

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Inactivation of Purified, Reconstituted Lauric Acid w-Hydroxylase by IO-UDYA-The binding of lauric acid and 10UDYA, a mechanism-based inhibitor of the microsomal w hydroxylase (31), to cytochrome P - 4 5 0 ~ ~ ~ Type I difyields ference spectra with Ks values, respectively, of 10.7 and 14.6 )IM (data not shown). The X value for lauric acid is compa, rable to thevalues of 1 and 18 p~ reported by Tamburini et 1 al. (2) and Hardwick et ul. (15),respectively. Inactivation studies were performed at 25 Cbecause preliminary experiments showed that lauric acid itself causes a significant loss of the w-hydroxylase activity at 37 C but not 25 C. Preincubation of P - 4 5 0 with~10-UDYA for various ~~ periods of time showed that approximately 45% of the whydroxylase activity is lost in a time-dependent manner (Fig. 5). The NADPH-dependent inactivation occurs rapidly but is followed by a slower NADPH-independent loss of activity, Loss of catalytic activity is observed whether cytochrome b5 is present or not (data not shown). A number of variations were tried to determine if the enzyme could be fully inactivated. These include varying the concentration of IO-UDYA from 0.05 to 1.0 mM, adding IO-UDYA prior to adding the dilauroylphosphatidylcholinein the reconstitution protocol, bubbling with oxygen to increase the oxygen supply, and increasing the cytochrome P-450 r e d u c ~ s e : c ~ c h r o mPe 450 ratio. In no instance did inactivation exceed 45-50% of the initial activity (not shown). The partial inactivation observed here contrasts with our earlier finding that most of the w-hydroxylase activity of clofibrate-induced rat liver microsomes is subject to inactivation by 10-UDYA (31). Incubation of the reconstituted enzyme with 10-UDYA under conditions that cause maximal loss of catalytic activity did not significantly decrease the enzyme chromophore measured by difference spectroscopy (Fig. 6). Some changes are seen in the spectrum due to the fact that the cytochrome b5 in the sample cuvette is reduced in the preincubation with
-

Portions of this paper (including Materials and Methods, part of Results, Scheme 1, and Figs. 1-4, 7, and 10) are presented in miniprint at the endof this paper, Miniprint is easily read with the aid of a standard magnifying glass. Full size photocopies are included in the microfilm edition of the Journal that is available from Waverly Press.

NADPH, but the same changes are observed whether 10UDYA i s present or not. Thus, the difference in the 420-nm region is not observed if cytochrome bs is omitted from the incubation. These results clearly demonstrate that loss of catalykic activity is not associated with destruction of the prosthetic heme group. Metabolism of IO-UDYA-[l-*C]10-UDYAwas synthesized by adding radiolabeled cyanide to the mesylate of 9decyn-1-01 and hydrolyzing the cyano function (Scheme 1). Incubation of radiolabeled 10-UDYA with the reconstituted cytochrome P - 4 5 0 ~ ~ system resulted in formation of 1,llundecandioic acid as the only detectable metabolite. The identity of the metabolite is based on co-elution of the radioactive material with an authentic standard after esterification with diazomethane (Fig. 8), The average yield of the diacid metabolite determined fromtwo independent experiments was 0.92 nmol of diacid/nmol P-450. The individual values for the two experiments were 1.1 and 0.75 nmol of diacid/ nmol P-450. Incubation for 10 rather than 5 min still only gave 0.92 nmol/nmol P-450 of the diacid metabolite. Production of the diacid is thus complete after 5 min even though the enzyme, as already noted, retains approximately 50% of its ability to hydroxylate lauric acid. If only 50% of the purified cytochrome P-450 enzyme is vulnerable to inactivation (see below), the partitionratio for the inactivation, defined as the number of metabolite molecules produced per inactivation event, i s approximately 2. ~ a d ~of P-450, by [I~ i ~ YA-Incubation ~ ~ e -CJIO- UD of radiolabeled 10-UDYA with reconstituted cytochrome P4 5 0 ~ followed by precipitation, filtration, and washing of ~~, the protein, led to binding of 0.3 nmol of fatty acid/nmol P450 (Table X). This value includes a correction for the binding observed in the absence of NADPH. When the incubation mixture was analyzed by passage through a Sephadex G-25 column rather than by filtration, the binding of radiolabel to the proteins of the reconstituted system was estimated to be 0.54 nmol of 10-UDYA/nmol P-450. When the proteins passed through a Sephadex G-25 column were further separated by affinity chromatography on 2,5-ADP-Sepharose 4B, approximately 0.48 nmol of label/nmol P-450 was retained by the cytochrome P-450. Onlya trace of label was associated with the cytochrome P-450 reductase fraction (0.04 nmol/ nmol of P-450). This trace is probably associated with the trace impurity of cytochrome P-450b detected by SDS-PAGE

18642

Lauric a-HydroxylaseHeme Acid Protein Versus Alkylation

0 08

FIG.6. Electronic absorption spectrum of cytochrome P - 4 5 o L A u reconstituted with 2 equivalents of cytochrome P-450 reductase, 2 equivalents of cytochrome b e , and
dilauroylphosphatidylcholine after (a)and before ( b ) incubation with 10-UDYA and NADPH. The peak a t approximately 425nm in the former spectrum is due to reduction of cytochrome b5 by NADPH. It is not present in the difference spectrum if cytochrome b5 is omitted from the reconstituted system. Essentially the same difference spectrum is obtained when the reconstituted system is incubated with NADPH but no 10-UDYA.

0 04

e 0
n
Q

VI

0.00

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- 0.04
wavelength (nm)

1
, 1 1 1 1 , 1 1 1 1 1 1 ,

20

28 36 time ( m ~ n l

44

FIG.8. HPLC analysis of the metabolites produced in incubations of [1-14C]UDYAwith reconstituted cytochrome P4 5 0 ~The radioactivity in 1-min eluent fractions is indicated by ~~. the Y-axis on the left. The cross-hatched area represents the counts added standards in each fraction. The absorbance a t 217 nm due to the is indicated by the axis on the right. Only the relevant part of the high pressure liquid chromatogram is shown. The structures of the materials represented by the two peaks in the chromatogram are indicated.

FIG.9. SDS-PAGE of the protein fractions obtained by 2,5-ADP-Sepharose 4B affinitychromatography of the reP ~~ constituted cytochrome - 4 5 0 system after incubation with [1-14C]UDYA. Lane 1, molecular weight standards; lane 2, the
incubation mixture before affinity chromatography; lane 3, the cytochrome P-450 reductase fraction; lane 4, the cytochrome P - 4 5 0 ~ ~ and cytochrome b5 fraction. The details of the experiment are given under Materials and Methods (Miniprint). The band behind the cytochrome P - 4 5 0 band is due to a contaminating isozyme that ~~~ does not contribute detectably to lauric acid hydroxylation.

in the cytochrome P-450 reductase fraction from the affinity column (Fig. 9). Cytochrome P-450 reductase is therefore not measurably labeled. SDS-PAGE of the radiolabeled protein coupled with exposure to x-ray film confirm that the label is concentrated in thecytochrome P-450 fraction (not shown)?
Preliminary SDS-PAGE/radiography studies were carried out by Dr. Angela Wilks.

The data indicates that approximately 0.5 mol of lahel/mol of cytochrome P-450 is bound if all the P-450 is involved in the reaction, or approximately 1mol of label/mol of P-450 if only 50% of the enzyme is involved. The fact that UDYA only causes loss of 40-50% of the lauric acid o-hydroxylase activity suggests that only roughly one-half of the enzyme is vulnerable to inactivation, so that inactivation is associated with binding of one molecule of inhibitor toeach molecule of inactivated enzyme.

Lauric Acid w-Hydroxylase Protein Versus Heme Alkylation

Properties of Inactivated Cytochrome P-450"The cytochrome P-45OLAwrecovered from incubations with UDYA is indistin~ishable SDS-PAGE from control cytochrome Pby 450. Direct comparison by differencespectroscopy of the enzyme before and after inactivation, however, provides evidence of some change in the active site. A reverse Type I difference spectrum with a maximum pe~-to-trough difference of 0.03 absorbance units is obtained when the spectrum of enzyme incubated with 10-UDYAand NADPH is recorded versus that of enzyme incubated without 10-UDYA or NADPH (not shown). This difference spectrum is obtained after passage of both enzymes through Sephadex G-25 to remove small molecules. No significant differences are seen in the spectra of enzyme from control incubations without either or both NADPH and 10-UDYA.

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of weak C-H bonds have met with some success.The maximum primary specificity so far achieved in a model system has been attained by substituting phenyl groups at the ortho positions of the four phenyl groups in meso-tetraphenylporphyrin (16). Theortho phenyl substituents form a steric barrier that forces substrates to approach the iron-bound activated oxygen viaa narrow opening directly above the iron (16). The primary selectivityof meso-tetraphenylporphyrinis changed from 0.017 to 0.24 by addition of the ortho-phenyl substituents (Table 11). The high preference for secondary C-H bonds observed with both m e ~ l l o p o ~ h y r systems in and cytochrome P-45&, and the modest shift in specificity caused by the addition of ortho-phenyl substituents tomesotetraphenylporphyrin, delineate the extraordinary reaction control that must be exerted by cytochrome P-4501,~~ and other w-hydroxylases to overcome the inherentpreference for DISCUSSION w-1-hydroxylation. The oxidation of 10-UDYAby cytochrome P-45oLAu yields Lauric acid hydroxylation is maximized by a 2:l ratio of 1,ll-undecandioicacid as the only detectable metabolite. Earcytochrome P-450 reductase to cytochrome P-45oLAu in the reconstituted system. The further addition of 1equivalent of lier studies have shownthat aryl acetylenes are enzymatically unamcytochrome bs increases 12-hydroxylation 2-3-fold without oxidized to 2-arylacetic acids (26), but this is the first biguous demonstration that alkylacetylenes are similarly detectably increasing 11-hydroxylation. NADPH consumption studies indicate that this acceleration is due to more oxidized to carboxylic acids.By analogy withthe mechanism of efficient coupling of NADPH consumption to 12-hydroxyl- established for oxidative metabolism the aryl acetylenes, it is likely that oxygen transfer to the terminal carbon results ation. The reaction stoichiometry is thus approximately 1mol in concomitant migration of the terminal hydrogen to the of hydroxylauric acidfmol NADPH in the presence of cytovicinal carbon. Addition of water to the ketene produced by chrome bs but only 0.5 mol of hydroxylauric acid/mol NADPH this oxidative shift yields the isolated diacid in itsabsence. Western blot immunoquantitation of cytochrome P - 4 5 0 ~ ~ ~ P I R"CH*"r=O 5R"CHH*C02H HO R-mH' shows that cytochrome P - 4 5 0 1 represents 1-2 and 16-30% ~ of the total hepatic cytochrome P-450 content of, respectively, The ketene metabolite, however, can react with nucleophiles control and clofibrate-induced Sprague-Dawley rats (Fig. 2). other than water and is therefore probably also responsible This is considerably lower than the22 and 54-67% estimated for the 10-UDYA-mediated inactivation of cytochrome Pby radial immunodiffusion for, respectively, control and clo- 4 5 0 ~ ~ Earlier studies with aryl and alkyl acetylenes have ". fibrate-induced Wistar rats (14). A close correlation exists shown that oxidation of the terminal acetylenic moiety by a between the clofibrate-induced increase in cytochrome P- cytochrome P-450 enzyme results in N-alkylation of its pros4 5 0 protein estimated from Western blots (-15-fold) and thetic heme group (26-28). The structures of the resulting ~ ~ ~ the increase in the rateof lauric acid hydroxylation (-14-17heme adducts indicate that N-alkylation involves addition of fold). The increase in protein concentration estimated by the activated oxygen to the internal carbon and a porphyrin radial immunodiffusion (-2-fold), in contrast, does not cor- nitrogen to the terminal carbon of the triple bond. In contrast, relate well with the increase in the catalytic activity. It is ketene formation requires addition of the activated oxygen to therefore likely that the values determined by radial immu- the terminal carbon. This relationship between the site of nodiffusion are too high due cross-reactionof the antibodies oxygen addition and the catalytic outcome explains why the to with other microsomal proteins. prosthetic hemegroup of cytochrome P h "& k % is notNLauric acid is oxidized much more rapidly by cytochrome alkylated during the catalytic turnover of 10-UDYA (Fig. 6). P - 4 5 0 than~cytochrome P-450b (Fig. 4). Furthermore, 11- Heme alkylation does not occur because the enzyme is con~~ and 12-hydroxylauricacids are produced in approximatelyan structed so as to specifically deliver the activated oxygen to 81ratio by cytochrome P-45h but a 1 5 7 ratio by cytochrome the terminal carbon. This leads uniquely to theketene interP - 4 5 0 ~ Statistical correction of the ratio of terminal to mediate and therefore to the diacid metabolite and protein ~~. internal lauric acid hy~oxylation to allow forthe presence of acylation rather than heme alkylation (Fig. 11). three terminal butonly twow-1 hydrogens providesan index The inactivation data indicates that something in theorder of the specificity of the enzyme for primary versus secondary of half of the lauric acid w-hydroxylaseactivity is resistantto C-H bonds. The primary selectivities thus obtained for inactivation by 10-UDYA (Fig. 5). One explanation for this cytochromes P - 4 5 0 ~ and P-45h are11.3 and 0.083, respec- is that a single enzymeis wounded rather than inactivated by ~tively. These valuesshow that the primary specificity of reaction with 10-UDYA so that it oxidizes lauric acid at a cytochrome P-45h in the oxidation of lauric acid is compa- reduced rate. The alternative explanation is that the enzyme rable to its primary specificityin the oxidation of simple preparation contains not one but two distinct lauric acid whydrocarbons (Table 11). Furthermore, cytochrome P-45h hydroxylases. The latter explanation better rationalizes the exhibits the same high preference for the oxidation of second- observation that formation of 1,ll-undecandioic from 10ary over primary C-H bonds that is characteristic of steri- UDYA ceases completely when the lauric acid hydroxylase cally unhindered metalloporphyrin systems (Table 11). The activity is maximally inactivated. This is most evident in the intrinsic preference for the oxidation of secondaryversus finding that 10-UDYAincreases the consumption of NADPH primary C-H bonds is consistent with the view that the ease above background levels only a very briefperiod, whereas for of hy~oxylationis inversely proportional to the carbon- NADPH consumption is elevated throughout the experiment hydrogen bond strength (51). Efforts to construct metallopor- when lauric acid is the substrate (Fig. 7). The finding that phyrins that override the intrinsic preference for the oxidation loss of approximately one-half of the catalytic activity is

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18644
-N /
Fc

Lauric Acid w-HydroxylaseProtein Versus Heme Alkylation

/

N PROTEIN MODIFICATION

R-C"CH

-c

II 0

FIG. 11. Mechanism proposed for the inactivation of lauric acid w-hydroxylase by IO-UDYA. Delivery of the activated oxygen to the terminal carbon (path a)leads to formation of the ketene that acylates the protein or adds water to give the observed 1,llundecandioic acid metabolite. Addition of oxygen to the internal carbon (path b ) is required for the formation of a prosthetic heme adduct and is not observed with cytochrome P - 4 5 0 ~ ~ .

FIG. 12. Schematic representation of the active site of lauric acid w-hydroxylase

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tional modification of a single isozyme. The partition ratio for the inactivation of cytochrome PTABLE I 4 5 0 ~defined as thenumber of moles of 10-UDYA oxidized ~~, Binding of radiolabeled IO-UDYA under catalytic turnover conditions per mole of inactivated enzyme, is exceptionally low. If allowto proteinsof the reconstituted cytochromeP-450 system
See "Materials and Methods" for details.

ance is made for the fact that only roughly one-half of the enzyme can be inactivated, each molecule of the vulnerable +NADPH -NADPH Difference enzyme appears to oxidize three molecules of 10-UDYA before nmol IO-UDYA/nmol P-450 it is inactivated, two of which appear as 1,ll-undecandioic Filter assay acid and one of which iscovalently bound to theprotein. The Run 1 0.40 0.12 0.28 inactivation reaction is not well coupled to utilization of Run 2 0.52 0.20 0.32 NADPH because the NADPH consumed in the short burst -P-450 0.04 0.03 0.01 of activity during which enzyme inactivation occurs indicates 0.03 [l-"C]UDYA only that approximately 20 mol ofNADPH areconsumed per mole Sephadex G-25 assay of enzyme that is inactivated (Fig. 7). The enzyme thus turns Complete system 0.37 0.91 0.54 over approximately 10 times per molecule of 10-UDYA that Sephadex G-25 plus it oxidizes. Nine molecules of NADPH are presumably conaffinity column sumed in uncoupled reactions that reduce molecular oxygen 0.66 0.18 0.48 P-450 P-450 reductase 0.06 0.02 0.04 to hydrogen peroxide or water (Ref. 52 and references therein) and only one in the actual oxidation of 10-UDYA. The efficiency of the inactivation of cytochrome P-450 by agents that TABLE I1 alkylate the prosthetic heme group is substantially lower than Hydroxylation regiospecificities for linear hydrocarbons that observed here. The partition ratios for substituted phenylacetylenes range from 38 for p-methylphenylacetylene to 4 Reference Substrate System" for p-nitrophenylacetylene (42), and the partition ratios for 16 MnTPP(0Ac) Heptane 0.034 simple olefins fall in the 100-300 range (28). 16 0.017 FeTPP(0Ac) Heptane The high efficiency of the inactivation reaction suggests 16 0.59 MnTTPPP(0Ac) Heptane that protein alkylation occurs before the ketene diffuses out 16 0.24 FeTTPPP(0Ac) Heptane of the active site. The increased polarity of the ketene metabHepatic microsomes olite makes it likely that it has a weaker affinity for the 0.17 20 Heptane Uninduced rat PB-induced rat 0.07 20 Heptane enzyme than does lauric acid (K, = 11 p ~ or) 10-UDYA (Ks BP-induced rat 20 0.03 Heptane = 14.6 p ~ )However, the concentration of the ketene metab. 0.03 21 Hexane Cytochrome P-450b olite if it diffuses quantitatively into the medium before it 51 0.04 Octane alkylates the protein cannot exceed 1 p ~ It. is unlikely that This work 0.08 Lauric acid the high specificity and efficiency can be reconciled with This work Cytochrome P-450" 1. 13 Lauric acid formation of the alkylating agent at concentrations substanThe porphyrin abbreviations used are: TPP, meso-tetraphenyltially below those of the probable K, value. porphyrin, and TTPPP, meso-tetra(2,4,6-triphenyl)phenylporphyrin. The active sites of the lauric acid w-hydroxylases must be PB, phenobarbital; BP, benzo[a]pyrene. Primary selectivity = (terminal hydroxylation/number of termi- highly structured in the vicinity of the activated oxygen to nal hydrogens)/(internal hydroxylation/number of accessible internal suppress the highly favored w-1-hydroxylation (Table 11). hydrogens). This reaction control is probably exerted, as suggested by experiments with model metalloporphyrin systems (161,by associated with covalent binding of one-half a molar equiva- structuring the active site so that only the terminal methyl lent of 10-UDYA (Table I) also argues strongly for the pres- group reaches the activated oxygen. The tolerance of the ence of two lauric acid hydroxylases, one that oxidizes 10- enzyme for fatty acids of somewhat different chain lengths UDYA and is inactivated by it, and one that simply does not suggests, furthermore, that the terminal methyl specificity is accept 10-UDYAas a substrate. The two w-hydroxylaseactiv- not governed by specific interactions of the protein with the ities could be due to two distinct isozymes or to post-transla- carboxyl group. Access to the oxygen is therefore probably

s:t?&

Lauric Acid w-HydroxylaseProtein Versus Heme Alkylation

controlled by steric constraints within the catalytic site, as illustrated schematically in Fig. 12. The structural features that favor w-hydroxylation of lauric acid also favor w-oxidation of 10-UDYA. Since oxygen addition to the terminal carbon is concerted with migration of the terminal hydrogen to thevicinal carbon (26), the w-specificity of the enzyme leads to exclusive formation of the ketene metabolite that probably acylates a nucleophile in the catalytic or substrate binding sites (Fig. 11). Conversely, the fact that the inactivation of cytochrome P-45b by phenylacetylene is quantitatively accounted for by heme N-alkylation even though it produces substantial amountsof phenylketene suggests that cytochrome P-45b does not have a suitable active site nucleophile (42). The active sites of the two lauric acid w-hydroxylases in our purified preparation differ markedly in that only one of the two enzymes is inactivatedby 10-UDYA.The observation that conversion of 10-UDYA to the diacid metabolite ceases when the vulnerable enzyme is inactivated indicates that the resistant enzyme accepts lauric acid but not 10-UDYA as a substrate. The extraordinary specificity of the resistant enzyme for the saturated terminusof lauric acid thus protects it from inactivation by 10-UDYA. Furthermore, neither isozyme appears to accept the terminal olefin as a substrate because (a)loss of epoxidase activity caused by the olefin is not accompanied by loss of lauric acid w-hydroxylase activity, and ( b ) inactivation of lauric acid w-hydroxylase by10-UDYA occurs very quickly butits inactivation of the epoxidase activity occurs relatively slowly. The epoxidation thus appears to be catalyzed by a trace contaminating isozyme not involved in the w-hydroxylation of lauric acid. The active sites of both lauric acid w-hydroxylases thus exclude the terminal olefin analogue and one of them also excludes the terminal acetylene analogue of lauric acid. A comparison of the active sites of these two enzymes should prove highly informative with respect to the factors that control substrate specificity in cytochrome P-450 catalysis.
Acknowledgments-We would like to thank Jennine Lunetta for excellent technical assistance in enzyme purification, Barbara Swanson and Mark Watanabe for obtaining the mass spectrometric data, and Dr. Angela Wilks for the preliminary SDS-PAGE/radiography results.
REFERENCES
1. Kupfer, D. (1980) Pharmacol. Ther. 11,469-496 2. Tamhurini, P. P., Masson, H. A., Bains, S. K., Makowski, R. J., Morris, B., and Gibson, G. G. (1984) Eur. J. Biochem. 139,235-246 Parkhill, L. K., Yasukochi, Y., Masters, B. S. S., Theoharides, 3. Okita, R. T., A. D., and Kupfer, D. (1981) J. Biol. Chem. 256,5961-5964 4. Needleman, P., Turk, J., Jakschik, B. A., Morrison, A. R., and Letlowith, J. B. (1986) A n m Reu. Biochem. 55,69-102 5 . Holm, K. A., and Kupfer, D. (1985) J. Biol. Chem. 260,2027-2030 6. Williams, D. E., Hale, S. E., Okita, R. T., and Masters, B. S. S. (1984) J. Bid. Chem. 259,14600-14608 7. Yamamoto, S., Kusunose, E., Ogita, K., Kaku, M., Ichihara, K., and Kusunose, M. (1984) J.Biochem. (Tokyo)96,593-603

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8. Ogita, K., Kusunose, E., Yamamoto, S., Ichihara, K., and Kusunose, M. (1983) Biochem. Int. 6,191-198 9. Kaku, M., Ichihara, K., Kusunose, E., Ogita, K., Yamamoto, S., Yano, L, and Kusunose, M. (1984) J. Biochem. (Tokyo)96,1883-1891 10. Shak, S., and Goldstein, I. M. (1985) J. Clin. Inuest. 7 6 , 1218-1228 11. Romano, M. C., Eckhardt, R. D., Bender, P. E., Leonard, T. B., Strauh, K. M., and Newton, J. F. (1987) J. Biol. Chern. 2 6 2 , 1590-1595 12. Brash, A.R., Jackson, E. K., Saggese, C. A., Lawson, J. A., Oates, J. A., and Fitzgerald, G. A. (1983) J. Pharmacol. Ex Ther. 226,78-87 13. Roberts, L. J., 11, Sweetman, B. J., and Oates, A. (1981) J. Biol. Chem. 256,8384-8393 14. Bains, S. K., Gardiner, S.M., Mannweiler, K., Gillett, D., and Gibson, G. G. (1985) Biochem. Pharmacol. 34,3221-3229 15. Hardwick, J. P., Song, B.-J., Huberman, E., and Gonzalez, F. J. (1987) J. Biol. Chem. 262,801-810 16. Cook, B. R., Reinert, T. J., and Suslick, K. S. (1986) J. Am. Chem. SOC. 108,7281-7286 17. Nappa, M. J., and Tolman, C. A. (1985) Inorg. Chern. 24,4711-4719 18. Khenkin, A., Koifman, O., Semeikin, A,, Shilov, A., and Shteinman, A. (1985) Tetrahedron Lett. 26,4247-4248 19. Fontecave, M., and Mansuy, D. (1984) Tetrahedron 40,4297-4311 20. Fmmmer, U., Ullrich, V., Staudinger, H., and Orrenius, S. (1972) Biochim. Biophys. Acta. 2 8 0 , 487-494 21. Morohashi, K., Sadano, H., Okada, Y., and Omura, T. (1983) J. Biochem. (Tokyo)93,413-419 22. Terelius, Y., and Ingelman-Sundbe~,M. (1986) Eur. J. Bioehem. 161, 303-308 23. Vatsis, K. P., Theoharides, A. D., Kupfer, D., and Coon, M. J. (1982) J. Biol. Chem. 257,11221-11229 24. Ortiz de Montellano, P. R. (1986) in Cytochrome P-450: Structure, Mechanism, and Biochemistry (Ortiz de Montellano, P. R., ed) pp. 217-272, Plenum Publishing Corp., New York 25. Kerr, J. A. (1966) Chem. Reu. 66,465-500 26. Ortiz de Montellano, P. R. (1985) in Bioactiuation of Foreign Compounds (Anders, M. W., ed) pp. 121-155, Academic Press, New York 27. Ortiz de Montellano, P. R., and Reich, N. 0. (1986) in Cytochrome P-450: Structure, Mechanism, and Biochemistry (Ortiz de Montellano, P. R., ed) pp. 273-314, Plenum Publishing Corp., New York 28. Ortiz de Montellano, P. R. (1988) in Progress in Drug Metabolism (Gibson, G. G., ed) Vof. 11, Taylor & Francis, Basingstoke, England, in press 29. Gan, L. S. L., Aceho, A. L., and Alworth, W. L. (1984) Biochemistry 2 3 , 3827-3836 30. Gan, L. S. L., Lu, J. Y. L., Hershkowitz, D. M., and Alworth, W. L. (1985) Biochem. Biophys. Res. Commun. 129,591-596 31. Ortiz de Montellano, P. R., and Reich, N. 0. (1984) J. Biol. Chem 2 5 9 ,

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4lRfL"lAl

32. Calacoh, C. A., and Ortiz de Montellano, P. R. (1986) Biochemistry 25,

~ I ~ L A 1I ~

_"_ __"

33. CaJacoh, C . A., Shephard, E. A., and Ortiz de Montellano, P. R. (1987) Fed. Proc. 46,1955 34. Chan, W. K.,CaJacob, C. A., and Ortiz de Montellano, P. R. (1988) FASEB J. 2 , A562 35. Macaulay, S. R. 11980) J. Org. Chm. 45,734-735 36. Valicenti, A. J., Pusch, F. J., and Holman, R. T.(1985) Lipids 20,234-242 37. Corey, E. J., and Suggs, J. W. (1975) Tetrahedron Lett. 2647-2650 38. Fieser, L. F., and Fieser, M. (1967) Reagents for Organic Synthesis, Vol. 1, pp. 135-139, John Wiley & Sons, New York 39. Fieser, L. F., and Goto, T.(1960) J. Am. Chem. SOC. 82,1693-1697 40. Shephard, E. A., Pike, S. F., Rahin, B. R., and Phillips, I. R. (1983) Anal. Biochem. 129,430-433 41. Waxman, D. J., and Walsh, C. (1982) J. Biol. Chem. 257,10446-10457 42. Komives, E. A., and Ortiz de Montellano, P. R. (1987) J. Biol. Chem. 2 6 2 , 9793-9802 43. Laemmli, U. K. (1970) Nature 227,680-685 44. Lowry, 0. H., Rosebmugh, N. J., Farr, A. L., and Randall, R. J. (1951) J. Bid. Chem. 193,265-275 45. Omura, T., and Sato, R. (1964) J. Bioi Chem. 239,2370-2378 46. Gorsky, L. D., and Coon, M. J. (1986) Drug Metab. Dispos. 14,89-95 47. Phillips, I. R., Shephard, E. A., Bayney, R. M., Pike, S. F., Rabin, B. R., Heath, R., and Carter, N. (1983) Bmhem. J. 212,55-64 48. Thomas, P. E., Reik, L. M., Ryan, D. E., and Levin, W. (1981) J. Bwl. Chem. 256,1044-1052 49. Towhin, H Staehelin, T., and Gordon, J. (1979) Proc. Natl. Acad. Sci. U. S. A. 76;'4350-4354 50. Hawkes, R., Niday, E., and Gordon, J. (1982) Anal. Biochern. 119, 1431 A7

_."

51. Jones, J. P., Korzekwa, K. R., Rettie, A. E., and Trager, W. Am. Chem. Soe. 108,7074-7078 52. Pompon, D. (1987) Btochemistry 26,6429-6435

F. (1986) J.

Continued on next page,

18646

Lauric Acid ~ - H y d ~ o xProtein VersusHeme A l k ~ ~ t i o n y~e

S ~ P P L E h ~ E ~ T A MATERIAL RY
to

THE CATALYTIC SITE OF RAT HEPATIC LAURIC ACID WHYDROXYLASE Protein v s Prosthetic Alkylation Heme in t hw - H y d r o x y l a t i oo f e n Acetylenic Acids Fatty Claire A. Calacob.William K. Chan. ElizabethShephsrd,and

Paul R. OrtizdeMontellano

M*uri.lr Glycerol (Gold labei), cyanogen bromide. l.8-diamin~lctanc. 10undecm-lol.andI.ll-undacamdioic acid were from Aldrich: NADPH. DETAPAC. DBAE-Scphacel, CM-Saphwse 68. CM-Scphadex C5O. Scpharose 4B. sodiumcholate. clofibrate were fo Sillnu; dilavroylphosphalidyIcholine WII rm c l o f i t i c r i d . and ctby1 from Serdlry Research Laboratories (Pon Huron. MI): Bio-Gel hydroxyl;lpatite. HTP Chsler resin, nnd horseradish peroxidase-conjugated goat anti-rabbit IgG were from Bie-R.d: 2:S"ADP Scpharore 4 8 was from Pharmacia. Emulgcn was kindly provided by KAO A t l u Corp. (Tokyo. Japan). [l-14Cllauric r i d (15.30 mCiImmol) and l14ClKCN (53 mCiimmo1) were obtained from Amenham. Unlabeled IO-UDYA was synthesized as previwsly described (31). Deionized. glass distilled water was used for all biological
work.
The stepthe of Synthesis of ( l - ~ 4 C 1 - t O - L ' a d ~ c y n o i eAcid. first in synthrsts radiolabeled IO-undecynoic (Scheme acid I). synthesis 9-dccyn-1-01, of was carricd out by themethod of Macaulay (3%. Sodiumhydnde (50% in 011. 9.48 g. 0.2 mol) was placed in a 5 0 0 mol two-neck romd bottom flzme.driod flask undcr nztrogen and was freed of mineral oil by washing hexane. with Thesolid was then suspended in 148 ml 1 of I . 3 d i ~ i urd othe mixturc was sttrred . 70C for 2 hr b c f w it was ~ ~ ~ 1.3. cmled in an ice bath. A solution of 3-dccyn-1-01 (3.9 g,25 mnol) in 79 ml of dipminopropane was then added dropwise with a dropping funnel the and resulting mixture w u stirred at 55OC overnightand Was then emled u) O' C in an ice bath. Aftcr concentrated HCI was addeddropwise to bring the pH IO approximately 3, the mixture wasextracted with diethyl ether the and combined ether extracts were washed water and saturatedaqueous NaCl. Drying over anhydrousMgSO4. sequentially with under vacuum yielded and distillation at a rotary evaporator. removal of the solvent 2.89 8 (19.3 mmol. 77%) of 94ecyn-I-of: IH NMR (CDCl31 3.63 11, 2 H. J = 6.2 Hz. CHzO-). 2.15 (m. 2 H. -CH2-C<-). 1.92 (1. I H. J = 2.6 H z C=CH). 1.49 (I. 2 H. . CH2CH2OH).and 1.34 ppm (I. IO H. methylenes: IR (ncat) 3353(hydroxyl).2114 (acetylene stretch). and 630 cm-1(acetylenebend). The mesylate of 9.decyn.l-01 was synthesized by the procedure of Valiccnti et ai. (36).A solution of 9.decyn-1.01 (19.3 mmol, 2.98 g) in I 5 ml of drypyridine was cooled to 0C in adryround bottom flask uodcr a nitrogen atmorphere.Mcthaneml) w u added by syrin8e the and solution was stirred for 30 sulfonyl chloride (7.5 mi" at O T and 60 min at r w m temperature before it was rrcmled to O*C. The sol~tton was acidifiedbydropwiseaddition of cold I G S aqueous HCI and was thenextracted with diethyl ether. The combinedether extracts wem washed with cold IO% HCI and then with raturzwt sodium bicaibonaa solution. Drying OYCI anhydrous sodium and by sulfate, removal of thesolvent on I rotary evaporator. purification packedwith230-400meshsilica gel with chromatography on a 1.5 x 14cmcolumn benzene PI the elution %oI~enf gave 4.09 g (17.6 mmol. 91.2%) of 9-dccyn-1-01 merylaur: IH NMR (CDCI3) 4.22 (1. 2 H. J = 6.2 Hz. -CH2OSOz-). 2.99 (I, 3 H. -0S02CHp). 2.16 (m, 2 H. -CHZC=C-), 1.93 (1. I H. I = 2.7 H I -C-dH). and 1.35 ppm (m. I 2 H. (acctylcnic CH). 2114 (acetylene stretch). 1171 and cm-I methylenes): IR (neat) 3287

KCN (0.0116 g. 1.1 mmol) war dissolved in mthanoi and was combined with the was carefully Il4C1KCN ( I mCi. 56 mCiimmo1) in a round bottom flask.Themethanol removed under if tiea am of nitrogen in a hood. A drying tube was attached the and KCN driedovernight by heating at 11O'C in an oil bath. The flask was then cooled and so as to place the internal contents the drying replaced a tube withcondenser fitted solution of 9-decyn-1-01 mesylate (0.2284 g. 0.98 undernitropen a atmosphere. A mmol) in 1.0 ml of dimelhylsulforide was added via syringe. The mixture was heated for 1.5 hr at 85C. Aftercooling 10 r w m tcmperatum. 15 ml of walcr was addedand ether. The combined extracts were warhcd the mixture was extracted with diethyl with 5 % sodium bicarbonate solution and were dried anhydrous over magnesium sulfate. Solvent removal at a rotary evaporator yielded 0.162 g (0.99 mmol. 100%) of 11~14C1-10-undecylnitnlc: i H NMR (CDCIJ) 2.33 (1. 2 H. 1 = 6.5 Hz, -CHZCN). 2.17 (m, 2 H. -CHzC=C-) m 1.93 (I 1 H. J = 2 6 Hz. -C=CH), and 1.37 ppm (m. 12 H, methylenes): IR (neat) 3296(C=CH).2246(C=N), and 2114cm-1(acetylene).
A solution of tl-~4C1-lO-undecynyinilriie(0.162 g. 0.99 mmol) and 0.6 g of KOH (approximately 10 mmol) in 2.5 ml of 95%ethanol and 1.0 ml of water was refluxed at 85Cfor24 hr. The mixture was then cooledand was acidified by dropwireaddition of 1 0 8 aqueous HCI. The mixture was extractedwithdiethyletherandthecombincd extracts were washed with water and with saturated aqueous NaCl solution before they were dried over anhydrous MgSO4 andlhe solvent rcmovsd a1 a rotary evaporator. The residue was purified by passage through a silica gel column (disposable pipette) preequilibrated with 8 0 2 0 petroleum ether:dicthyl and ether eluted diethyl with ether. eluate. The refrystrllirtd from petroleum ether, gave 0.154 g (0.84 mmol. 85%) 11-14CI-10-undecynoic of acid: 43-44-C. m.p. mixed meltmg point The with authentic sample 43-44C: NMR identical to authentic sample. specific ~ictwity of the radiolabeled material was 5.08 x 103 cpminmol. The same synthetic scheme w3s employed a second time, starting with 17 umol labeled KCN and 33 "mol cold KCN, to make [I-14CIUDYA with a specificradioactivity of 2.85 I 104 cpmimoi.

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Synthesis of l l - ~ ~ C l - 1 1 ~ D o d e c e n oAcid. i c Methane sulfonyl-chloride (7.5 ml. 97 mmol) was added by syringe to a stirred Solution of I0.undecen.l-ol (3.8 ml. 19 mmol) in dry pyridine(14 mi) ai 0C (nitrogen atmosphere!. Thc mixtum was stirred at 0C for 30 min. at 25C for I hr. and finally at O'C for 40 min before cold 10% HZ304 was added dropwine IO bring the pH to about 2. Themixture WBE then extractcd with diethyl ether the snd combined ether fractsons. afterdryingwithanhydrousNa2SO4. were concentrated on a rotary evaporator. resulting The ycllow liquid (15.1 mmol) was Shown by NMR to Consisl of IO-undecylenyl-1-mesylate c ~ " t ~ m i n ~ t ~ da with Small a m o ~ n t of the starling alcohol. A s o l u t m of 1O~undecyIenyl.l-m~sylale6 6 mp. 0.27 mmol) in dry ( dimethylsulfoxide (0.5 ml plus0.5 a mi wash) was injected into a capped scintillal~m vial containing a nixed solution of Kl4CN (1 mCi. 53 mCiimmo1) and 18 mg of unlabeled KCN in 0.5 r l of dry dtmethyl-sulfoxide. The solution was stirred at 110C n Sor 1.5 hr and was thenallowed to cool to room rempaturc before slowly adding 2 ml of water. The mixture was extracted five times with diethyl and ether the combined elher extracts were (NaZSOO), dried filtered, and evaporated to dryness under D Stream of nitrogen.The residual ycllow oil was shown by NMR and F n R analysis to be I I-undecylenyl-I-nitrile. KOH (0.28 g). ethanol (Iml), and wafer (0.5 ml)wereadded to the labeled 11. undecylenyl~l.nirrile the and mixture war stirred under "itmgm at 85C for 29 hr. The mixture was then coolcd and 10% HZSO4 was added until the solution turned cloudy about (pH 2). The mirture was extracted diethyl with ethor. the ether extiacls Were dned(Ns2SO4). and the solvent allowed to evaporate in ahood.The residual yellowliquid(18.3 mg. 34.2%yield) had an NMR spectrum identical to that of an nuthentic sample of 1 I-dodecenoic wid.

(Sa).

oxidation was carried out by the The Synthesis of 12-Oxododecanoic Acid. acid g. 10 procedure of Corey and Suggr (37). A suspension of 12-hydroxylauric (2.15 mnrol) in 25 mi ofmethylenechlorrdcand of ~ y , i d i ~ i " m~ h l ~ ~ (3.25 h g, ~IS ~ m ~ mmol) in 20 r l of methylene n chloride combined the were and resulting mixture was sinred at room lcmperature under nitrogen forhr, 3 Diethyl ether (200 ml) was added the and black reactioo mixture was filtered. filtered The ether fraction was rotary evaporator. and the residue passed through Slorisil. the ether was removed on a was ptrcif?ed on a 1.S x 14 em column packedwith230-400 mesh silica g d (Aldrich) eluted with diethyl ether (0.83 g. 3.9 mmol. 40%): IH NMR 9.76 (I, IH. J = 2 Hz, CHO). 2.35 (m, 4 11, -CH2C02H and -CH2CHO). 1.60 (m, 2 H. - C H ~ C H ~ C O Z H ) , 1.29 ppm (s. and IC H. methylenes), IR ino~ol)1708(COZH), 1736 cm-f (CHO). The baric procedure was Synthesis of i1,lZ.Epoxydode~anoir Acid. that of FiesetandFieser(38). A solution of 11-dodecenoic acid (1.98 g. 10 mmol) in I S ml at merhylenechloride was added dropwire at 0% under niirogcn to a solution of mchloropcrbenroic acid (1.95 g. 11.3 mmol) in 25 ml of methylcnc chloride. The mixture was stirred 2 hrs a t TOQm temperaturebefore IO ml of a IO% sodium sulfite Solution was added to decompose excess peroxide. mixture The was then extracted thrirc with 5 % sodium bicarbonate soiutmn, once with water. and twice with saturated aqueous NaCl sul*tion. Theorganiclayer vas dzied over anhydrous r d i u m sulfate. the selvcnt removed at P rotary evaporator. and the residue purified by chromatography on a 1.5 x 14 em column packed 230-400 with mesh silxa gel elutedfirstwith 9:l ~ " ~ ~ " e : d i ~ etherand then with thyl diethyl t her alone. This yielded 0.58 g (2.7 mrnol. 30%) of the desiredepoxide: IH NMR (CDC13) 2.90 (m. I H, intcmal eporide proton). 2.74 It. I I). I = S Hz. cisepoxide proton). 2.48 (m, I H, trans eporidc proton). 2.35 (1. 2 H. 1 = 7 Hz. RCH2Ctr2). and 1.30 ppm (m, 16 H.mcthylcnes): IR (nujol) 1687 (carboxyl). 850 cm-1 (epoxide).

the of 11.14CI-10-undecynorc actd Scheme I.Synthetic sequence for preparation (DAP = 1.3~di~mmopropanei. similar A iequence was used In [he syntllesrs of 1 1 ~ 1 4 C l I I-dodecenotc a a d .

Lauric Acid w-Hydroxylase Protein VersusHeme Alkylation

11,lL.Dihydrox~dodPranoic Acid. The procedure o f Ficrer and Got0 Was employed (391. To a s o l ~ t i o n the epoxide (0.43 g. 2 mmoll in 10 ml of of tetrahydrofuran war added 0.8 ml of 30% aqueous perchlortc acid and the mixture was nllawed 10 stand for 6.5 hr ar room temperature under a nitrogen atmosphere. Water ( l o ml) was then added and the mixture was extracted thrice with 25 ml of d i a h y l ether. The combined organic layers were washed with water and satmated NaCl solution and were drlcd over anhydrous sodium rulfatc. Solvent removal at a rotary evaporator loliowcd by ~ ~ ~ ~ y ~ t ~ l l i ~ hexane:ethyi acetate provided from I:[ ~ t i ~ " 0 I g (0.4 mmol. 20%) of the pure diol: I H NMR (D20) 3.19 (m, 3 H. RCHOHCHZOH), 1.91 (1. Z 1, J = 7 Hz. RCH2COz-I. and 0.89 ppm (m. 16 H. methylenes): I R (nujol) 3374 (hydroxyl) and 1694 cm.1 (carboxyl).
Animals. Sprague~Dau,ley male rats (180-200 g) were induced with clofibrate i n In early expermeno, rats wexe fed wrth Normal Piolein Test Rat Diet (ICN Biochcm!cals, Cleveland) supplemented with 0.4% cloiibric acid for 14 days. In later experiments. the ran were inpcted intraperitoneally srhyl with clofibrate at a dose of 400 mglKg once 3 day for three days. The animals were used to prepare the enzyme 24 hr after the last dose of ethylclofibrate. There were no noticeable differences io the enzyme obtained from the m s induced by the two procedures.
two waya.

18647

phosphate (pH 7.4) buffer containing 20% glycerol and 250 uM DETAPAC.14C-Sodlun1 laurate (0.55 mM) in dimethylsulioox~de.and NADPH 12.0 mMi. Afterinrubaling for 5 min at 2SC, the reactions were quenched by adding 10% sulfurlc m d 2nd the products were analyzed as usu3I.

Anlibodier. Antibodies 10 purifted lauric acid w.hydroxylase were raised in two male New Zealand While rabbits using the protocol reported by Phill~pret 81. ( 4 7 ) . Each rabbit was injected in the subrcvpular space with 50 ug of the antigen mtxed l : t ( v i " ) with Freund's adjuvant. Three weeks later. the rabbits were ~njected wlth 25 ug of the antigen similarlymired will>Freund's adjuvant. Five weeks later. the rabhits were given a 12.5 ug booster injection of the antigen and 7 drys later blood w m withdrawn from the marginal ear Yein and was allowed lo clot at room rempei:llure for 30 min. The c l o t w3s broken. stored at 20C overnight, and centrifuged at 10000 6 for I S min ar 4C. The antiserum was decanted and stored i n aliyuolr a1 -20C. The boorter and antiserum collection steps were repeated a i two month m t e r v ~ l s The presence of antibodies specific l o lauric o-hydroxylase acid u a s conflrmcd hy Ouchterlony double-immenodlffuiion analysis as described by Thomas ct d l . 14x1. Thc antihodier were found 10 react w t h the o.hydrorylase hut not ulrh P450h o r P45lk hy Western blot analysis.

P450 reductase was prepared by the procedure of Enzymes. Cytochrome Shephard et 21. (40). Cytochrome h5 and cytochrome P450b were purified from the livers of phenabarbital-pretreated rats by the procedure of Waxman and Walsh (41). The ~mplemenlation of there prcparotmnr and the quality of the proteins obtained i o thts laboratory have heen reported (42). Lauric acid w-hydroxylase war purified from the I~YPIS clofibrate-induced Sprdgue-Dziwley male rats (180 200 g) essenually as of derfiibed by Tamhurini et al. (2). From 20@0 mg of microsomes (20 rats) 10-50 "mol o i purified enzyme (approximately 1% yield) was obtained. The punty of the enzymes w x assessed by SDS-PAGE In a 7.5% gel by the procedure of Laemmli 143).

Western blot analysis of the con~entrationsof lauric acid o.hydroxylnsr in uninducsd and induced rat h v e r microsomes was carried out by the procedure provided with Bio~Radkits based on thar u i Towbin et 91. ( 4 9 ) . Sample? of puziiird I m n c acid o.hydroxylase rangmg from 3-10 pmol. mmosomCs from umnduced f a t livers (E0 and 40 p g total protein). and microbomcs from clofthrate-mduced iitl h e r ug total protem). were run together on 7 5% SDS gels The proteins were I15 and 7.5 in transferred to nitr~cellul~se a B w R a d T~ansblor apparatus operated at 1011 mA overmght followed by 200 mA far I hr.Non-rpcciiic protein hinding SNCE %ere blocked with gelatin. Goat anta-rabbu IgG con~ugaled w t h horseradish pero.vdase was employed as the second anrihody and 4-chloro-1-naphthol was used as the pcioridvse substrate (50). The protems stained by this procedure wcic quantmted by densilometry. I h e concenrratiun~ of lauric acid o-hydroxylase were derermmed wirh respect to the linear sectmns of Ihr atrndard curves gentrated wlth authentic o hydroxylase.
10-Undecynoie .4eid Metabolitcs. Cytochrome P450LAco

Downloaded from www.jbc.org at University of California, San Francisco on March 7, 2009

( I nmoliml).

Arrays. Prolein ~oncentration3 were measured by the method of Lowry with bovine .;erum albumin as ?he rtandard 144). The lauric acid o-hydroxylase activity w a s mcarured by ]>quid scmti11at~on counting of the metabolites produced from radiolabeled lauric acid a f m their separation by HPLC B I described earlier (31). Cytochrome P.450 was quantitated hy dtfference apecrrorcopy of the reduced. COsaturated versus reduced enzyme on an Arniiico DW-2a instrument. The 450-490 nm rhrorbmce difference and an a t i n c t $ o n coefficient of 91,000 M - I Em-1 w e r e employed far Lhls purpose (45). To determme the abiolute position of the Soret band uf P45OLAo. cytochrome P-450 (0.85 nmoliml) in IOU mM potassium phosphate buffer. pH 7.4. containing 20% glycerol and 251) u M DETAPAC was reduced with dilhronite and the ramplc was split into two ~uvettes. After the baielinc was recorded from 400 10 500 nm at 0.2 nmirec. carbon monoxlde was bubbled into the sample cell and ihe difference silect~um w3a recorded.

cytochrome P450 reductase (2 nmol/ml). 11-14CCIUDYA (0.6 mM1. and dilruroylphoaphat~dylcholine (40 ug/ml) were incubated 5 min 81 2 5 T and wfre then taken up in 100 mM potassium phosphate (pH 7 4 ) buffer containing 2 0 1 glycerol and 250 pM DET, 'AC. Cytochrome bg 12 nmollmll was added (final vOlUmC 2 ml) and the mixture incubated 5 mi" a i 25'C before NADPH ( 1 m M ) UBI added to initiate the re~ct~on. After 5 mln 81 25C. the mixture was quenched wllh 0 8 ml of 1 0 % HZSOI and unlabeled IO-UDYA (2 pwol) and l.lt-underanedio~c aced (2 pmd) were rddcd. The mixture was extracted with diethyl ether 13 L 4 ml) and the combtned extrim\ were passed through a rmdl pad of riltca gel m a disposable p&pe!te. concenlratcd 10 a v o l ~ m eof 2 ml under a stream of nitrogen. combined wrth2 ml of ethercrl diazomethane, and allowed to stand for 45 mi" a1 room temperature. The s ~ l v e n tw;n then removed under a stream of nitrogen rn a hood and was replaced with SOU pl of 1 : l ~c~tonitrllc:waler.A 100 pl alquot oi the resullmg solution was analyrcd by hlgh pressure liquid ~ h , - ~ t * g ~ ~ ~ ony a I 5 x 4.6 cm Dupont Zorhax ODS 5 um ClX revcrsc h phase column eluted with 1:l ( v i v ) ace~mltrile:water as a flow rate of 0.8 mllmin. The column effluent war monitored at 217 n m wtth a vaimble wavelength detector.
Dodrcenoie Acid Metabolites. [I-14CIII-dodeccnoir acid was mcuhotcd with reconstituted cytochrome P450LAw as desfrlbed above for IO4JDYA. l n t c r n d standards (12-oxododecanoic acrd, 11.12.epoxydodecsnoic acid, 1.2 dihydroxydodecanoic acid) were then addcd und the lncubat~on mixtures were ektracted twice withdlelhyl rrher. 'The erfiacts were faltered through rma!l ulic'' gel columns beiore diazomethrne ( 3 ml) was added to each vial and the v i a l ? were c.gpped and allowed 10 sand 45 mln a: room temperature. The iolvent and excess dzaromethane were then remowd under 3 ilreem of nmosen and 0 . 5 mi of 31:lY methanol.watcr was added to each vial. The resulting SOIUIIOIIE were analyzed by IIPLC using the s y ~ t e ~ l d conditions used for the analysis of lauric m acid tneraholito.

Enzyme Reconstitution and lncubalian Conditions. Pariiied lauric acid 0hydroxylase was reconstituted cytochrome wtth P450 reductase and. except where indicated. with cytochrome h5 by the gtnersl procedure of Gorsky and Coon (461. The conccnlr3rmns given ~n thmi and the subsequent procedures are rhose in the final incubelton mixtures. Typically. iufficicnt 100 mM potassium phosphate (ptl 7.4) huffercontaining 20% glyccrol and 250 aM DETAPAC wan added to a mixture of cytochrome P450 (0.5 nmoliml). cytochiame P450 reductase ( I nmoliml), and dil3uroylphosphat~dyl-~h~l~"~ (40 )~glml) 10 make the final incubation volume 0.45 nil. This mjxture war Incubated for 5 mi" a1 25C before adding cytochrome b5 ( 1 nmaltml). After an add%tzand5 min at 25C. 5 ul of dimcihylsulfooxide or a similar v0Iumc of d~melhyliulfox~de contalnmg the substrate or inactivat~ng agent was added. NADPH (0.5 mhll was then added to iniliate [he reaction and thc mixiwe was incubated for 1 ~ 5 (inactivatmn min experiments) or 6 mi" (activity assays) at 2 S T . In thc inactivation expeamentr. a 100 pl aliquot of the tncubalion mixture was then transferred to a prewarmed mlxiure o i d~lauroylphospha~idyrchoiine (I? ppiml). NADPH (0.5 mM). and 14C~Iauric acid (0.55 m M ) in 100 mM potassium phorphote (pH 7.31 buffer conraining 20% glycerol End 250 uM DETAPAC ( f i n d voliime 1.0 mi). The mixture wds m u b a t e d :it Z S T for the mdicated period of time (6 mi" unless otheiwise indicated) before the reactmn was quenched by adding 10% ("1") aqueous s u l l u r ~sod. Lauric acid and XIS metabol~ter were then extracted and analyzed by high presrure Isquid ~ h ~ ~ m a t o ~ ~ a p h y as reporled previously (31). Incubation o i the enzyme for various time perlodr in the absence of substrater or inhibitors followcd by dilution and a s a y as described hcre showed [hac the activity assay employed in inhihttor studics IF Isnear

Protein Labeling with 11-14CIUDYA. These exper!menrr were carried out by two dlfferent methods. onc involving filtration through Whstman filler paper and lhe othcr column chromatography on Sephadcr G-25. The incubation pioceduie. whrch was the same m both cases. involved Incubation of cytochrome P450LAw (I nmnllml).
cytochrome P450 reductase (2 nmollml). II-14CIUDYA ( 0 6 mM) and dihuroylphosphalidyl~h~line pgiml) for 5 mln at 2S'C prior 10 adding 100 mM i40 potassium phosphate (pH 7.4) buffer containing 20% glycerol and 250 uM DETAPAC. Cytochrome bg (2 nmollml) was then added and thc mixture was incubated a further 5 mi" at 25C before NADPH (1.0 mM) was added 10 mitivie the reacuon. Afier 3 nlm at 25C. the incubation was put on ice and 4 ml of cold 25% trcchloroacet~actd WAS added and thc mixture was allowed 10 sand for 5 m m The precipitated prolem war collected by Suction filtration i h r w g h 2.4 cm diameter Whatman G R B f?lrcr disc). The dscr were rmsed cold 25% trichloroacetic acid (4 K 1.5 ml). cold Rh tr~chlnroaccuc rcd (8 x 1.5 mil. and acetone (4 x 1.5 mi). Conrrvl experimcnrr indicated t h ~ txhe laheled hpid was not retained by the filters after thir procedure.

N A D P H Consumption Experiments. Lauric acid w.hydroxylase (0.5 nmaliml), cytochrome Pa50 reductase 11 nmollml), Sllauroyl-phospharidyl~h~line 4 0 pgiml). ( and inhibitor (0.3 mM1 ~n dimethylsulfox~dc or 0.55 m M lauric acid in the same volumf of dimethyl,ulforide were Incubated for 5 min at 2 5 T before adding sufficient 100 m M potassium phosphate (pH 7.41 buffer containing 20% glycerol and 250 pM DETAPAC s o that the finalmubarion volume would bc 0.5 ml. Cytochrome hg (I nmolimi) was then added and the mixture was incubated for r further 5 min at 25C before NADPH (0.3 m M ) was added to initiate cacalylic tumover. The absorbance at 340 nm was followedcontinuo~sly for I S min on a Hewlett Packard 8450A diode array IJViVIS spectrophotometer against a reference cuvctlc that contained d ~ l u u t o y l p h o s p h a u d ~ l ~ h ~(40 = l i ~ pglml). 0.3 m M dimethylsulfoxide, 20% glycerol, and 250 uM DETAPAC an 100 mM polasslum phosphale (pH 7.4) buffer. The absorbance changes were converted to nmolcs usmg the cxtinclion coeffic~entfor NADPH of 6.22 x 103 M.1 cnt-1.

I n the alternative procedure. the clrmplete reaction mixture was incubated for 5 mi". The incubation mixture war then put on ice until i t could k loaded on a 1 I 35 cm column of Sephadcx G-25(fine grade) that had been swollen and preequihbrated with 50 mM Triris-acetate buffer (pH 7.41 containing 20% glycerol, 0.1 mM EDTA. and 0.5% (w/v) sodium cholate. The column was eluted with the Same buffer system and 7 mi" fractions werr collected. The absorbance o f each fraction was measured at 412 nm to determine the P450 cantent and the sample was subjected to liqutd scintil1mon counnng.

Catalytic Dependence on R atio of Cytochrome P450 Reductase to Cytochrome P450. A standard I ml mcubarion war set up by incubating cytochrome P450 (0.2 nmolimll. one of the following ~ o n c e n t ~ a l i o n s cytochrome P450 reducurc of (0. 0.1, 0.2, 0.4. 0.7. 1.2. 2.0 nmollml). and dilauroylphosphatidylcholine (40 pglml) for 10 min at Z S T . T o ihts was added the requisite amount of 100 m M potassium

In some instances. 5 min fractions were collected from the Stphadex G-25 column and those fractions containing the hemoprotein were pooled. A 25 pl aliquot wits taken for SDS-PAGE and the rest war loaded onto a 1 x 3 cm (2 ml volume) column o 2 ' s f ADP~Scpharore4 8 that had been prcequilibrated with 50 mM Trir-acetate p H 7.4 containing 20% glycerol. 0.1 m M EDTA, and 0.5% sodium cholate. The cytochrome P450 and hs were eiurcd with the same lris buffer containing, in addition, 0.75 M KC].The KC1 u.31 added to minimme ionic inlerhclion~ between the hemoprotem and flavoprotein. The reductase was then eluted wish the same T m bufieer containing 5 mM NADP+. A 30 pl sample of each fraction w85 rcmovcd for SDS-PAGE andthe remainder was subjected to laquid sciotillation counting.

18648

Lauric Acid w-Hydroxylase Protein Versus Heme Alkylation

Labeling. A system containing by Covalent Spectroscopic Changer Caused P 4 5 f l L A w (1.33 nmollml). cytochrome P450 reductase (2.66 nmollml). I O - U D Y A (0.6 mM). dllluroylphorphatldylcholinc (40 pglml). 20%glyceroland 250 p M D E T A P A Ci n I00 m M potasuum phosphrle buffer (pH 7.4) was reconrtiwted as usual (10131 volume 0.75 ml). N A D P H ( 1 . 0 m M ) was then added and the mixtureIncubated 5 mi" at 25 'C cooled m ~ c eand put through a Sephadex G-25 column as already before i t W B ~ dercrtbed. The cnzymc contaming fractions were poalcd and concentrated w i t h Ccntricon fsltcrs (10.000 MW-cutoff). Similar experiments were carried out wlthaut NADPH.withoutIO-UDYA.andwithoutbothIO-UDYA and NADPH.The absorbance at 412 nm of il 0.5 ml volume o f each sample was adjusted wtth Trirbufferuntil all the v d u c s (c.g. the protein conccntratmnr) were the same The sample ablained from the incubation wkthaut bothIO-UDYA and N A D P H was then balanced against i t r c l ff r o m 350 lo 500 nm on the Hewlctt Packard dmde orray spectrophotomerer and the absorbance difference spectrum rccorded. sample The cuvelte was then replaced in lurn by each of thcolhcr lhrce sampler (-NADPH. +IO-UDYA: +NADPH. -IO-UDYA: and +NADPH.+IO-UDYA) and the absorbance difference spectrum recorded.

and

RESULTS

Purificationnd haracterization a C of L a u r i c c i d - H y d r o x y l a s e . A o P~SOLA,. the cytochrome P450 thdt catalyzes w-hydroxylatmn of lauric actd. was purlfted from the livers of claf~hrarc-prclreatcdrats by the procedure Tamburmi of et ill. 12). The enzyme thus ohtamed had a spcclfic content that ranged from 7 to 12 nmol l'450lmg protein. speetftc The conlcnt reported Tamburml by et 31. is 12.8 nmol P4501mg protetn ( 2 ) and by Hardwnck et al. 16-18 nmol P450lmg protem ( I S ) . SDSPAGE of the enzyme (Figure I ) shows that our enzyme preparation Contains two almost unresolved bands that run more slowly cytochrome than P450b. I n slightly less well purified preparations a well resolved lower band observed is (e+. Figure 9 ) . The protein responsible for thin lower band t s not detectably involved i n lauric acid hydroxylatmn because the same resulls are ohtamed in its presence or absence. The absorbance maximum of the reduccd. carbon mononde.haund enzyme. a s reportcd by Tamburini et PI.. IS at 452 n m (2).

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\
enzyme at 37'C in the The production of I I - and 12-hydrorylrurie acids by the abscnce of cytochrome b5 was 8 - 1 1 nmollnmol P4501mm. I n the presence of cylochromc bg. the catalytic activity was 18-20 nmollnmol P450lmin at 37C. There values were obtained at a 2:lratio of cytochrome P450 reductase to cytochrome P450 because this is the m m i m u m ratto required maximum for actiwly (no1 shown). Tamburini et al. report an aetlvity of 43.0 nmol productlnmol P450lmin in the abscnce of cytochrome and bg Hardwick et PI. a value o f 18.5 nmollnmol P4501min in the presence of cytochrome b5 ( I S ) . The 12-hydroxylated lauric acld i n the presence of cytochrome b5 acco~ntsfor 95.99% of the product. Il-Hydroxylauricacidis usually P minor. often barely detectable. metabolite. proportions The of the two products i n parallel incubations and with without cytochrome indicate cytochrome bg that bg stimulates formationof thc 12-hydroxylatedproductby a factor of 2 IO 3 but has no measurable cffect on produelmn of the Il-hydroxylated product. Tamburini et al reported their that preparmon gave at best B 6:l ratio of the 12- Il-hydrorylatcd to products (2). Evidence that the 0 - 1 hydroxylation primarily is catalyzed by a contaminating isozyme i n the o.hydroxylsse preparationprovided is by the observation the that w-hydroxylase aclivity declines much faster on storage than thc 0 - 1 hydroxylase activity. different Two enzyme prepnralmns stored at -79C for 1 R and 7 months. respectively. retamed 15.20% of their 0 - 1 hydroxylase activity but only approximately 0.1% of them o-hydroxylase activity. Well aged preparauonr consequently oxidize lauric slowly acid to a 1:6 mixture of the ro-and w - l products.

mo IgG

mmol e 5 0

Figure 3. lnhlhitionoflauric acrd w - and 0 - 1 hydroxylation bymllhodlcr to cytochrome P45flLAo. hydroxylase The acIIviles were mcacurcd under the rtmddrd conditmnsin the presence of increnbing amounts of I g G from cytochromeP450LAwimmunized rabbits. The experimental details arc given tn Malcrlaln and Mcthodr. Western blot analyslr of the content of immunodetectableP45flLAw tn mlcro\ome% from uninduced and cloftbrate-induced rats. bared on a standard curve ertabllrhed wlth increasing amounts of the authemic enzyme (Figure 2). establish that ,he protun 1s Induced several fold cloflbrrle. mmunochemlcal by The merhod indicale? that P 4 5 0 L A W is present i n uninduced and induced mtcrosomes at COnCenlrationS of IX-33 and 322-605 pmollmg protein. respectively. Analysis of the lo111 amount of cytochrome P450 i n the Same microsomes dtffcrence by spcctroscopy gtver values for the uninduced mdueed and microsomes of 1.34 and 2.03 nmallmg. rerpecllvely. These values indlcate cytochrome that P 4 5 0 L A o represents only 1.3-2 5% of !he lmnl P450 content of unmduccd microsomes and 16.30% o f the enzyme in clafihrateInduced mmosomes. This IR-fold induction a t the protem l c v e l agrecr WCII wuh thc 17-fold induction nn the o-hydroxylaseactivities of the same mmosomes. The 0 - 1 hydroxylase acttvity. as expected t f itisprimarily catalyzed b y a different isozyme. is only elevated 2.5-fold by clofibrate trealment.

R e g i o s p e c i f i c i l y of L a u r i c A c i d H y d r o x y l a t i ob y n P150b and P ~ S O L A ~ . The II- and 12-hydroxylation of lauric acld reconstituted by cytochromes P450h and P 4 5 0 L A o has been quantitatively measured. As shown tn Ftgurc 4. P45flb gwer rhc II. and 12-hydroxylated metabolltcs i n a ratio approximately The of 8:I. prnmrry aelecrivily the for process. defined a s (12-hydroxylaura1ell I-hydrorylaurate) x (number of II-hydrogenr)/(number of 12-hydrogens). ls 0.083. In ConIrast. P45flLAw giver the I I - and 12-hydroxylated metabohter ~n approximately a 1:17 ratlo. Thc primary selectivity for the reaction catalyzed P450LAw lhcrelore by is approxtmatcly 11.3. The two enzymesdifferintheirpnmaryselectivityby a factor o f about 136

l m m u n o q u a n t i t a t i a n of H e p a l iL a u r iA c ih y d r o x y l a s e . n t i b o d i c r c c d A raised 10 purified cymchromc P450LAw in rabhils recogmzcd the two poorly rcsolved hands o f the purlfied enzyme preparation (figure 21 but did not crossreact w i t h cytochrome P45flb or P450e. Addlng ~nereasmg amounts o f the anlibody to tncubattonr of lauric with acld thc reconrtrtuted cytochrome P 4 5 0 r~ r l~ m~ r c v e d % . y e as cxpected. a concentration-dcpcndent inhihilion of the w - h y d r o x y l a t i o n reactlo" (Rgure 3). The 0 - 1 hydroxylation IS also inhibited but not wtlh !he same conccntrmnn dependence (now that the actud magnitude of the w - l hydroxylatmn 8s much lowcr than that for the o-hydroxylation). antibody The at a mncenlratmn of 25 mp. IgGInmol P450 depresses the w-hydroxylase acllvity by almost 90%.

P450b

p450LAw

Figure 4. Comparison of the formation of 1 1 - and 12-hydroxylauric acids by cytochromes P450b andP450LAo.Thesolid bars on the left are the values for 1 1 hydroxylation and the crosshatched bars on the r i g h t the values far 12-hydroxylation

Lauric Acid ~ - H y ~ r o ~ y l ~ e VersusHeme A l ~ l a t i ~ n Protein

The specificacttvity denoted by each bar is given above it I n units of nmol producllnmol enzyme. The ram of oxidanon of lauric acid by P450b and P450LAm are also qrlte different. Under our incubation conditions. the rates of formation of ihc 11- and 1 2 ~ hydroxylauric actds by cytochrome P45ob were 0.48 and 0.06 nmollnmul P450imln. rcspectively. corresponding The valuer for P 4 5 0 ~ ~ 0 0.84 and 19.21 nmollnmol were P450imm. respectively. The net rate of oxidation ai lauric acid was therefoore 0.24 nmollnmollmm by P 4 j o b and 20.05 nmollnrnollmln by P 4 5 0 ~ ~ o Interaction o f Cytochrome P 4 5 0 ~ ~ ~ with If-Dadecenoic Arid. KO lime dependent loss of lauric hydroxylase acid activlly or chromophore loss 1s detected when reconstituted cytochrome P 4 5 o L A o is incubared wtlh 11-dcdecenoii acld and NADPH (not shown). Radiolabeled 11-dodecenoic acid is, nevertheless. eniymati~ally oxidized to a 10:I mixture of 11,12-epoxydodecanoic acid and 12-oxododecano1c (Figure IO). Control experiments suggest that the diol arises bynmplc chemwal hydrolysis of the spoxrde during the incubation and isolation procedures but the aldehyde appears to be an enzymaric product. Epoxidat*on levels off after npproximately S mi" of incubation even though latlric hydrarylatton acid i s not atteneatcd. Preincubation of the reconstituted enzyme with IO-UDYA ICSUI~S m e In t dependent inactivation of the cpoxidation react~on. but inaetivat~on IS not complete after IO mi" whenas macrivation oi lauric acid o - h y d r o x y l ~ i o nby IO-1JDYA is e~sentialiy complete after I min (not shown).

N A D P H Canrumplion by Cytochrome P41iOl.A" and Partially P$SOLAo.No ;onsumption of NADPH is detected bymonitoring the absorbance at 340 nm i n incubations of NADPH without lauric acid. cytwhrome P ~ S O L A cytochrome P450 reductase, and cylochrome hs. NADPH consumpllon ls ~, detected when cytochrome P450 reductase 1s present and is ~ncrsmenrallyenhanced by the sequentml addition of cytochrome P 4 5 0 L A o and cytochrome b j (no1 shown). NADPH consumption is doubled If the enzyme concentrations are doubled. Thus. 81 25 oC. the rates of NADPH consumption are 4 . 3 4 and 6.77 nmollmin, respe;rwely, wtlh 0.5 and 1.0 nmollml of cytochrome P450. Addition of lauric acid 10 the complete sys:em markedly increases NADPH consumption. The itnear rate observed over the first 300 ~ e c mdicatcs that NADPH i s consumed at a tale of 1 I nmoiimininmul P410 (Figure 7). Product formallon in (he same period i s S nmolimininmol P4S0 i n the absence of cytochrome h5 and 10 " m ~ I l ~ i ~ i ~ m " 1 in i t s presence. P450 Approximrtely I nmol of NADPH i s therefore consumed per nmol of product i n the presence of cytochrome hg whereas in i t s absence the consumprion of NADPH i s ~ p p ~ ~ ~ i m2~ nmollnmol of product The primary cifect of cytochrome bs IS t e l y therefore 10 mrease the efficlency of the reaction rather than LO increme the rate of
Inactivated oxygen activation.

"

In ~ o n l ~ a s NADPH consumption i s onlyrlrghtlyscimulrted t. when 1O.UDYA is added 10 lhe incubation mixture. The change caused by IO-UDYA OCCUTS within the f m t 100 seconds. after whtch NADPH consumption i s roughly the same w t h and without IO-UDYA. The rapid hut small increment 10 NADPH con5umpson caused by IO-UDYA approxima!ely doubles if the enzyme concentrat~mis doublcd. The burst $"crease i n NADPH consumption caused by addltton of IO~UDYA I S approxlm~tely 20 "mol of NADPH per nmd of cytochrome P450. In view of the fact that apptortmately 2 molecules of 10-UDYA are oxidized per imctivation event (see below). one of which hinds covnlcntly to rhe enzyme. i t appears that 10 nmol of NADPH are consumed per molecule of IO-UDYA t h u is oxidized. The con?umption of NADPH and the activation of oxygen are therefore much less efficicntly coupled 10 the ox1dmon of IO-UDYA than lauric acid. Thc fale of the oxygen m the c a t r l y t ~ ccycle5 that do out result in hydroxylation of IO-UDYA IS not known. If oxygen is reduced $0 the level of hydrogen peroxide. the ~nzyrneturns ovm nine T ~ C before i t succeeds in J oxidizing rhe acetylenic substrate. If oxygen IS reduced 10 water. the enzyme goes through half as many futde catalytic cycles. ! contrast. there are essentially no fuule n cacalpc cycles when lauricacid is nxidzzed I" the presence of cytochromehs.

7
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o
10

20

30

Time (min)
Figure 10. Famation of the Il,l2-eporide. 12-07.0. and 11,12diol metabolites of 11-dalecsnaicacid(DDEA) as a function of the timc o f incubation of the radiolabeled olefin with the reconstituted cytoehromc PdXJLAo system. l l , l Z - ~ ~ ~ ~ y d a l e c = (l). acid ~ ~ i ~ l2-onododccanoic acid and il.12dihydroxy-dodecanoicacid ( 0 ) .

(e)

Figure 7. Consumption of NADPH by 9 system composed of cyrochromt P 4 5 0 L ~ ~ . cytochrome P4S0 reductarc. and dilauroyl~phorphrlidylchollne ( a ) i n the a h x n c e of added ruhrtrates, (b) i n thc presence of IO-UDYA. and IC] a the pceseoce of lauric n acld. NADPH conrump..m isgiven PS the change I" the rhrorbance of the cofmor 3% 340 nm. The experimental procedures are glven ~n Methods and Mater~alr.