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THE OURNALF BIOLOOICALHEMISTRY0 1988by The American Society for Biochemistry a nd Molecular Biology, Inc Vol. 263, No. 35, Issue of December 15. pp. 186411-18649,1988Printed in U . S . A .

The Catalytic Site of Rat Hepatic LauricAcid o-HydroxylasePROTEIN VERSUS PROSTHETICHEME ALKYLATION IN THE w-HYDROXYLATIO~ OF ACETY~ENICFATTY ACIDS*

(Received for publication, May 16, 1988)

Claire A. CaJacobS, WilliamK.ChanS, Elizabeth Shephardg, and PaulR.Ortiz deM o n t e l l ~ o S ~From the ~ ~ e p a r t ~ n tf~ ~ r ~ e u t i c a ~hemistry, School of Pharmacy, and Liver Center, University ofCalifornia,Sun Francisco, California94143 and the WDemrtmentof ~ ~ ~ ~ r n i s t ~ ,n i v e r ~ ~ ~ollege, h n do n WC1E 6BT,nited Kingdom

Cytochrome P - 4 5 0 ~ ~ -urified from clofibrate-in-duced rat liver oxidizes lauric acid to 11- and 12-hydroxydodecanoic acid in approximate~y1: 7 atioat a rate of 20 nmolJnmol P-450Jmin. In contrast,cytochrome P-450b oxidizes lauric acid much moreslowly (0.5 nmol/nmol P-4FiO/min) to an 8:l mixtureof the same metabolites, Western blot analysis indi-cates that P - 4 5 0 ~ ~ ~ccounts for 1-2 and 16-30%,respectively, of the total cytochrome P-450 in unin-duced and clofibrate-induced rat liver. Cytochrome b6increases the fficiency of w-hydroxylation but not thera te of catalytic turnover. Incubation of the enzymewith 10-undecynoic acid (10-UDYA) results inloss ofapproximately 45% f the enzymatic activity butnoneof the enzyme chromophore. Approximately 1 mol of1,I -undecandioic acid is produced per mole of inac-tivated enzyme. This extraordinary inactivation effi-ciency is confirmed by N A D P H consumption studies.Aproximately 0.5 equivalents of label are covalentlybound to the enzyme when it is incubated with *C-labeled IO-UDYA. 11-Dodecenoic acid appears not tobe a substrate forytochrome P-450LAa but is oxidized,presumably by a contaminating isozyme, to a 1O: lmixture of 11,12-epoxydodecanoic acid and 12-oxo-dodecanoic acid. The results suggest the presence oftwo closely related P - 4 5 0 ~ ~nzymes, only one ofwhich issusceptible to inactivation by IO-UDYA. Theyalso indicate that cytochrome P - 4 5 0 ~ ~ ~as a highlystructured active site that sterically suppresses W-1-hydroxylation in order to deliver the oxygen to thethermodynamically disfavored terminal carbon. Pro-tein rathe r than heme alkylation follows from hisreaction regiospecificity.

A number of the known isozymes of cytochrome P-450 aredesigned to specifically w-hydroxylate longor medium lengthfat ty acids (1-4) as well as the structurally related prostaglan-dins (1,5-9), leukotrienes (10, ll), rostacyclins ( E ) , andthromboxanes (13). The number, detailed specificities, and~ h y s ~ o l o ~ c a ~oles of these w-hydroxylases remain ambiguous,* Support for this research was provided by National Insti tutes ofHealth Gran t GM 25515, the Cancer Research Campaign and theMedical Research Council of GreatBritain, and NorthAtlanticTreaty Organization Travel Grant 86/0789. The core facilities of theUniversity of California Liver Centerare supported by NationalInstitutes of Health Grant P-30 AM 26743. Th e costs of publicationof this article were defrayed in part hy the payment of page charges.This article must therefore be hereby marked ~ V e r ~ i s e ~ e R t naccordance with 18 U.S.C. Section 1734 solely o indicate this fact.1[ To whom correspondence should be addressed: School of Phar-macy, S-926, University of California, San Francisco, CA 94143-0446.

but it is clear from the available evidence that distinct iso-zymes catalyze the different w-hydroxylations.The lauric acidw-hydroxylases from rat liver (2) and pig kidney (3) , and theprostaglandin w-hydroxylases from rabbit liver ( 5 ) , lung (6,71, kidney (8),and intestine 9) have been extensively purified,albeit not n all cases to homogeneity. The lauric acid w -hydroxylase preparation of Tamburini et al. (2) was recentlyreported by Hardwick et al. (15) to contain two proteins. Thelatter investigators have isolated a cDNA clone hat codes forone of the two proteins, have sequenced it, and have demon-strated that it s a lauric acid w-hydroxylase by expressing itin catalytically active form in yeast. Quantitation of thehepatic lauric acid w-hydroxylase by radial immunodiffusionled Bains et al. (14) to suggest that 22 and 57 % of the totalcytochrome P-450 in the liver, respectively, of uninduced andclofibrate-induced rats is the lauric acid w-hydroxylase.The fatty acid w-hydroxylases are particularly interestingfrom a mechanistic point of view because they oxidize thethermodynamically disfavored terminal methyl group. Modelstudies with hypervalent oxometalloporphyrins show that ox-idation of the hydrocarbon methylenes is highly favored overoxidation of the terminalmethyl group (16-19). This inherentreactivity difference results in preferential w-1-hydroxylationof hydrocarbon chains by relatively nonspecific cytochromeP-450 isozymes ( e g . rat P-45&, rabbit P-450~~2)20-23).Preferential hydroxylation of the methylene groups is pre-dicted by the now favored hydrogen radical abstraction mech-anism (reviewed in Ref. 24) if one considers that the C--Hbond strength decreases in the order primary > secondary >tertiary. The bond dissociation energies for removal of ahydrogen to give the methyl, isopropyl, and t-butyl radicalsare thus, respectively, 98.0, 94.5, and 91.0 kcallmol (25). w-Hydroxylases must therefore override the inherent pecificityof the catalytic species for the weaker C-H bond.Hepatic cytochrome P-450 isozymes oxidize terminal acet-ylenes (RC=CH) to ketenes (RCH==C=O) hat add water togive acetic acid derivatives (RCH2C02H) (26). The cyto-chrome P-450 isozymes involved in the reaction are simulta-neously inactivated (27, 28). In most instances, inactivationis due to N-alkylation of the prosthetic heme group of theenzyme by the catalytically activated acetylene. The heme N -alkyl group obtained in the oxidation of R - C e H i s invar-iably NCH2COR. The possibility that acetylenes are alsooxidized to species that react with the protein matrix issuggested by the disparity between the decrease in the spec-

The abbreviations used are: heme, iron protoporphyrin Ix re-gardless of the iron oxidation and ligation states; SDS-PAGE, poly-acrylamide gel electrophoresis in the presence of sodium dodecylsulfate; DETAPAC, diethylenetriaminepentaacetic cid; 10-UDYA,10-undecynoic acid; HPLC, high pressure liquid chromatography.18640

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Lauric Acid o-HydroxyZase Protein Versus Heme A ~ k ~ l u ~ i o ~ 18641troscopically measured content of cytochrome P-450 and theloss of aryl hydrocarbon hydroxylase activity observed byGanet al. (29,30) in i n~ba t ionsf liver microsomes with ethinyl-substituted polycyclic aromatic hydrocarbons.

We have demonstrated that insertion of a terminal triplebond into the hydrocarbon chain produces fatty acid ana-logues that are highly selective mechanism-based inactivatorsof the fatty acid w-hydroxylases (31, 32). Inactivation of therat liver lauric acid w-hydroxylases by hese agents has beenfound to result in negligible oss of the cytochrome P-450chromophore. This can be explained either by the presence ofa very low titer of the w-hydroxylases or by inactivation via amechanism that does not cause chromophore destruction. TOclarify this question and to investigate the mechanism bywhich the enzyme promotes the intrinsically more difficult o-hydroxylation, we have purified lauric acid o-hydroxylase,quantitated it by Western blotting procedures, identified theproducts formed on oxidation of terminally unsaturated fattyacids, and investigated the mechanism of its inactivation bya radiolabeled acetylenic inhibitor. A part of this work hasbeen reported in preliminary form (33,34).

MATERIALS A ND METHODS*RESULTS

Inactivation of Purified, Reconstituted Lauric Acid w-Hy-droxylase by IO-UDYA-The binding of lauric acid and 10-UDYA, a mechanism-based inhibitor of the microsomal w -hydroxylase (31), to cytochrome P - 4 5 0 ~ ~ ~ields Type I dif-ference spectra with K s values, respectively, of 10.7 and 14.6)IM (data not shown). The X,value for lauric acid is compa-rable to thevalues of 11and 18p~ reported by Tamburini etal. (2) and Hardwick et ul. (15),respectively.Inactivation studies were performed at 25 C ecause pre-liminary experiments showed that lauric acid itself causes asignificant loss of the w-hydroxylase activity a t 37 C but not25 C.Preincubation of P - 4 5 0 ~ ~ ~ith 10-UDYA for variousperiods of time showed that approximately 45% of the w -hydroxylase activity is lost in a time-dependent manner (Fig.5). The NADPH-dependent inactivation occurs rapidly but isfollowed by a slower NADPH-independent loss of activity,Loss of catalytic activity is observed whether cytochrome b5is present or not (data not shown). A number of variationswere tried to determine if the enzyme could be fully inacti-vated. These include varying the concentration of IO-UDYAfrom 0.05 to 1.0 mM, adding IO-UDYA prior to adding thedilauroylphosphatidylcholine n the reconstitution protocol,bubbling with oxygen to increase the oxygen supply, andincreasing the cytochrome P-450 reduc~se:c~chrome-450 ratio. In no instance did inactivation exceed 45-50%of the initial activity (not shown). The partial inactivationobserved here contrasts with our earlier finding that most ofthe w-hydroxylase activity of clofibrate-induced rat liver mi-crosomes is subject to inactivation by 10-UDYA (31).

Incubation of the reconstituted enzyme with 10-UDYAunder conditions tha t cause maximal loss of catalytic activitydid not significantly decrease the enzyme chromophore meas-ured by difference spectroscopy (Fig. 6). Some changes areseen in the spectrum due to the fact that the cytochrome b5in the sample cuvette is reduced in the preincubation withPortions of this paper (including Materials and Methods, artof Results, Scheme 1,and Figs. 1-4, 7, and 10) are presented inminiprint at the endof this paper, Miniprint is easily read with theaid of a standard magnifyinglass. Full size photocopies are included

in the microfilm edition of the Journal that is available from WaverlyPress.

-

I 2 3 4 5tlme (mln)

FIG. 5. Inactivation of purified, reconstituted cytochromeP-450Lh by preincubation with 10-UDYA in the presenceand absence of NADPH. The loss of lauric acid a-hydroxylaseactivity observed by preincub ationwith 10-UDYA n the presence ofNADPH has not been corrected for he loss observed in the absenceof NAD PH. The experimental details are given n Materials andMethods (Miniprint).Little loss of activity is observed if the enzy meis preincubated without10-UDYA.NADPH, but the same changes are observed whether 10-UDYA i s present or not. Thus, the difference in the 420-nmregion is not observed if cytochrome bs is omitted from theincubation. These results clearly demonstrate that loss ofcatalykic activity is not associated with destruction of theprosthetic heme group.Metabolism of IO-UDYA-[l-*C]10-UDYAwas synthe-sized by adding radiolabeled cyanide to the mesylate of 9-decyn-1-01 and hydrolyzing the cyano function (Scheme 1).Incubation of radiolabeled 10-UDYA with the reconstitutedcytochrome P- 4 5 0 ~ ~ ystem resulted in formation of 1 , l l-undecandioic acid as he only detectable metabolite. Theidentity of the metabolite is based on co-elution of the radio-active material with an authentic standard aftersterificationwith diazomethane (Fig. 8) , The average yield of the diacidmetabolite determined from wo independent experimentswas 0.92 nmol of diacid/nmol P-450. The individual valuesfor the two experiments were 1.1 and 0.75 nmol of diacid/nmol P-450. Incubation for 10 rather than 5 min still onlygave 0.92 nmol/nmol P-450 of the diacid metabolite. Produc-tion of the diacid is thus complete after 5 min even thoughthe enzyme, as already noted, retains approximately 50% ofits ability to hydroxylate lauric acid. If only 50% of thepurified cytochrome P-450 enzyme is vulnerable to inactiva-tion (see below), the partition atio for the inactivation,defined as the number of metabolite molecules produced perinactivation event, i s approximately 2.

~ a d ~ ~ ~ e ~ i ~f P-450, by [I -CJIO- UD YA-Incubationof radiolabeled 10-UDYA with reconstituted cytochrome P-4 5 0 ~ ~ ~ ,ollowed by precipitation, filtration, and washing ofthe protein, led to binding of 0.3 nmol of fatty acid/nmol P-450 (Table X) . This value includes a correction for the bindingobserved in the absence of NADPH. When the incubationmixture was analyzed by passage through a Sephadex G-25column rather than by filtration, the binding of radiolabel tothe proteins of the reconstituted system was estimated to be0.54 nmol of 10-UDYA/nmolP-450. When the proteinspassed through a Sephadex G-25 column were further sepa-rated b y affinity chromatography on 2,5-ADP-Sepharose4B,approximately0.48 nmol of label/nmol P-450 was retainedby the cytochrome P-450. Onlya trace of label was associatedwith the cytochrome P-450 reductase fraction (0.04 nmol/nmol of P-450). This trace is probably associated with thetrace impurity of cytochrome P-450b detected by SDS-PAGE

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18642 Lauriccid-Hydroxylaseroteinersusem elkylation

FIG. .Electronicbsorptionspectrum of cytochrome P - 4 5 o L A ureconstituted with 2 equivalents ofcytochrome P-450 reductase, 2equivalents of cytochrome b e , anddilauroylphosphatidylcholineafter (a) nd before (b) incubationwith 10-UDYA and NADPH. Thepeak at approximately 425nm in theformer spectrum is due to reduction ofcytochrome b5 by NADPH. It is not pre-sent in the difference spectrum if cyto-chrome b5 is omitted from the reconsti-tuted system. Essentially the same dif-ference spectrum is obtained when thereconstituted system is incubated withNADPH but no 10-UDYA.

0 08

0 04

Eenm0VIQ

0.00

- 0.04

, 1 1 1 1 , 1 1 1 1 1 1 ,20 28 36 44t i m e ( m ~ n lFIG. . HPLC analysis of the metabolites produced in incu-bations of [1-14C]UDYAwith reconstituted cytochrome P-4 5 0 ~ ~ ~ .he radioactivity in 1-min eluent fractions is indicated bythe Y-axis on the left. The cross-hatched area represents the countsin each fraction. The absorbance at 21 7 nm due to the dded standardsis indicated by the axis on the right. Only the relevant par t of thehigh pressure liquid chromatogram is shown. The structures of thematerials represented by the two peaks n the chromatogram areindicated.

in the cytochrome P-450 reductase fraction from the affinitycolumn (Fig. 9). Cytochrome P-450 reductase is therefore notmeasurably labeled. SDS-PAGE of the radiolabeled proteincoupled with exposure to x-ray film confirm that the abel isconcentrated in thecytochrome P-450 fraction (not shown)?Preliminary SDS-PAGE/radiography studies were carried out byDr. Angela Wilks.

wavelength (nm)

1 2 3 4FIG. . SDS-PAGE of the protein fractions obtained by2,5-ADP-Sepharose 4B affinitychromatography of the re-constituted cytochrome - 4 5 0 ~ ~ystem after incubation wi th[1-14C]UDYA. Lane 1 , molecular weight standards; lane 2, theincubation mixture before affinity chromatography; lane 3, the cyto-chrome P-450 reductase fraction; lane 4 , the cytochrome P-450~~-and cytochrome b5 fraction. The details of the experiment are givenunder Materials and Methods (Miniprint). The band behind thecytochrome P - 4 5 0 ~ ~ ~and is due to a contaminating isozyme thatdoes not contribute detectably to lauric acid hydroxylation.

The data indicates that approximately 0.5 mol of lahel/molof cytochrome P-450 is bound if all the P-450 is involved inthe reaction, or approximately 1mol of label/mol of P-450 ifonly 50% of the enzyme is involved. The fac t that UDYAonly causes loss of 40-50% of the lauric acid o-hydroxylaseactivity suggests tha t only roughly one-half of th e enzyme isvulnerable to inactivation, so that inactivation is associatedwith binding of one molecule of inhibitor toeach molecule ofinactivated enzyme.

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Lauric Acid w -Hydroxylase Prote in Versus Heme Alkylation 18643Properties of Inactivated Cytochrome P-450"The cyto-chrome P-45OLAw ecovered from incubations with UDYA is

indist in~ishable y SDS-PAGE from control cytochrome P-450. Direct comparison by differencespectroscopy of theenzyme before and after inactivation, however, provides evi-dence of some change in the active site. A reverse Type Idifference spectrum with a maximum pe~-to-trough iffer-ence of 0.03 absorbance units is obtained when the spectrumof enzyme incubated with 10-UDYA and NADPH is recordedversus tha t of enzyme incubated without 10-UDYA orNADPH (not shown). This difference spectrum is obtainedafter passage of both enzymes through Sephadex G-25 toremove small molecules. No significant differences are seenin the spectra of enzyme from control incubations withouteither or both NADPH and 10-UDYA.

DISCUSSIONLauric acid hydroxylation is maximized by a 2: l ratio ofcytochrome P-450 reductase to cytochrome P-45oLAu in the

reconstituted system. The further addition of 1equivalent ofcytochrome bs increases 12-hydroxylation 2-3-fold withoutdetectably increasing 11-hydroxylation. NADPH consump-tion studies indicate that this acceleration is due to moreefficient coupling of NADPH consumption to 12-hydroxyl-ation. The reaction stoichiometryis thus approximately1molof hydroxylauric acidfmol NADPH in the presence of cyto-chromebs but only 0.5 mol of hydroxylauric acid/mol NADPHin itsabsence.Western blot immunoquantitation of cytochrome P - 4 5 0 ~ ~ ~shows that cytochrome P- 4 5 0 1 ~epresents 1-2 and 16-30%of the total hepatic cytochrome P-450 ontent of, respectively,control and clofibrate-induced Sprague-Dawley rats (Fig. 2).This is considerably lower than the22 and 54-67% estimatedby radial immunodiffusion for, respectively, control and clo-fibrate-induced Wistar rats (14). A close correlation existsbetween the clofibrate-induced increase in cytochrome P-4 5 0 ~ ~ ~rotein estimated from Western blots (-15-fold) andthe increase in the rateof lauric acid hydroxylation (-14-17-fold). The increase in protein concentration estimated byradial immunodiffusion (-2-fold), in contrast, does not cor-relate well with the increase in the catalytic activity. It istherefore likely that the values determined by radial immu-nodiffusion are too high due o cross-reaction of the antibodieswith other microsomal proteins.Lauric acid is oxidized much more rapidly by cytochromeP - 4 5 0 ~ ~ ~han cytochrome P-450b (Fig. 4). Furthermore, 11-and 12-hydroxylauric cids are produced in approximatelyan81 ratio by cytochrome P-45h but a 157 atio by cytochromeP - 4 5 0 ~ ~ ~ .tatistical correction of the ratio of terminal tointernal lauric acid hy~ox yla tio n tollow for he presence ofthree terminal but nly twow-1 hydrogens providesan indexof the specificity of the enzyme for primary versus secondaryC-H bonds. The primary selectivities thus obtained forcytochromes P- 4 5 0 ~~ -nd P-45h are11.3 and 0.083, respec-tively. These valuesshow that the primary specificity ofcytochrome P-45 h in the oxidation of lauric acid is compa-rable to ts primary specificity n the oxidation of simplehydrocarbons (Table 11). Furthermore, cytochrome P-45hexhibits the same high preference for the oxidation of second-ary over primary C-H bonds that is characteristic of steri-cally unhindered metalloporphyrin systems (Table 11). Theintrinsic preference for the oxidation of secondaryversusprimary C-H bonds is consistent with the view that the aseof hy~oxylation s inversely proportional to the carbon-hydrogen bond strength (51). Efforts to construct metallopor-phyrins that override the intrinsic preference for the oxidation

of weak C-H bonds have met with some success.The maxi-mum primary specificity so far achieved in a model systemhas been attained by substituting phenyl groups at the orthopositions of the four phenyl groups in meso-tetraphenylpor-phyrin (16). Theortho phenyl substituents form a stericbarrier that forces substrates to approach the iron-boundactivated oxygen viaa narrow opening directly above the iron(16). The primary selectivity of meso-tetraphenylporphyrinschanged from 0.017 to 0.24 by addition of the ortho-phenylsubstituents (Table 11). The high preference for secondaryC-H bonds observed with both m e ~ l l o p o ~ h y r i nystemsand cytochrome P-45&, and the modest shift in specificitycaused by the addition of ortho-phenyl substituents tomeso-tetraphenylporphyrin, delineate the extraordinary reactioncontrol that must be exerted by cytochrome P-4501,~~ andother w-hydroxylases to overcome the inherentpreference forw-1-hydroxylation.The oxidation of 10-UDYA by cytochrome P-45oLAu yields1,ll-undecandioic acid as the nly detectable metabolite. Ear-lier studies have shown ha t aryl acetylenes are enzymaticallyoxidized to 2-arylacetic acids (26), but this is the first nam-biguous demonstration that alkylacetylenes are similarlyoxidized to carboxylic acids.By analogy with he mechanismestablished for oxidative metabolism f the aryl acetylenes, itis likely tha t oxygen transfer to the terminal carbon resultsin concomitant migration of the terminal hydrogen to thevicinal carbon. Addition of water to the ketene produced bythis oxidative shift yields the isolated diacid

R - m H ' - "CH*"r=O 5 "CHH*C02HI H OThe ketene metabolite, however, can react with nucleophilesother than water and is therefore probably also responsiblefor the 10-UDYA-mediated inactivation of cytochrome P-450~ ~ " .arlier studies with aryl and alkyl acetylenes haveshown that oxidation of the terminal acetylenic moiety by acytochrome P-450enzyme results in N-alkylation of its pros-thetic heme group (26-28). The structures of the resultingheme adducts indicate that N-alkylation involves addition ofthe activated oxygen to the internal carbon and a porphyrinnitrogen to theerminal carbon of the triple bond. In contrast,ketene formation requires addition of the activated oxygen tothe terminal carbon. This relationship between the site ofoxygen addition and the catalytic outcome explains why theprosthetic hemegroup of cytochrome Pk%h& is notN-alkylated during the catalytic turnover of 10-UDYA (Fig. 6).Heme alkylation does not occur because the enzyme is con-structed so as to specifically deliver the activated oxygen tothe terminal carbon. This leads uniquely to theketene inter-mediate and therefore to the diacid metabolite and proteinacylation rather than heme alkylation (Fig.11).The inactivation data indicates that something in theorderof half of the lauric acid w-hydroxylase ctivity is resistant oinactivation by 10-UDYA (Fig. 5). One explanation for thisis tha t a single enzyme s wounded rather than inactivated byreaction with 10-UDYA so that it oxidizes lauric acid at areduced rate. The alternative explanation is that the enzymepreparation contains not one but two distinct lauric acid w -hydroxylases. The latter explanation better rationalizes theobservation that formation of 1,ll-undecandioic from 10-UDYA ceases completely when the lauric acid hydroxylaseactivity is maximally inactivated. This is most evident in thefinding that 10-UDYA ncreases the consumption of NADPHabove background levelsor only a very briefperiod, whereasNADPH consumption is elevated throughout the experimentwhen lauric acid is the substrate (Fig. 7). The finding thatloss of approximately one-half of the catalytic activity is

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18644N/ Fc /N

R-C"CH

Lauric Acid w-HydroxylaseProtein Versus Heme Alkylation0

PROTEINMODIFICATION

R

-cII0

FIG. 11. Mechanism proposed for the inactivation of lauricacid w-hydroxylase by IO-UDYA. Delivery of the activated oxy-gen to the terminal carbon ( p a t h a ) eads to formation of the ketenetha t acylates the protein or adds water to give the observed 1,ll-undecandioic acid metabolite. Addition of oxygen to he internalcarbon ( p a th b ) is required for the formation of a prosthetic hemeadduct and is not observed with cytochrome P - 4 5 0 ~ ~ .TABLE

Binding of radiolabeled IO-UDYA under catalytic turnover conditionsto proteinsof the reconstituted cytochromeP-450 systemSee "Materials and Methods" for details.

+NADPHNADPHifferencenmol IO-UDYA/nmolP-450

Filter assayRun 1 0.40 0.12 0.28Run 2 0.52 0.20 0.32[l-"C]UDYA only 0.03-P-450 0.04 0.03 0.01

Sephadex G-25 assaySephadex G-25 plusaffinity column

Complete system 0.91.37 0.54

P-450 0.66 0.18 0.48P-450 reductase 0.06 0.02 0.04

TABLE1Hydroxylation regiospecificities for linear hydrocarbons

System" Subs trate s:t?& eferenceMnTPP(0Ac)FeTPP(0Ac)MnTTPPP(0Ac)FeTTPPP(0Ac)Hepatic microsomesUninduced ratPB-induced ratBP-induced ratCytochrome P-450b

Cytochrome P-450"

HeptaneHeptaneHeptaneHeptaneHeptaneHeptaneHeptaneHexaneOctaneLauric acidLauric acid

0.0340.0170.590.240.170.070.030.030.040.0811.3

161616162020202151This workThis work

The porphyrin abbreviations used are: TPP, meso-tetraphenyl-porphyrin, and TTP PP,eso-tetra(2,4,6-triphenyl)phenylporphyrin.PB, phenobarbital; BP, benzo[a]pyrene.Primary selectivity = (terminal hydroxylation/number of termi-nal hydrogens)/(internal hydroxylation/number of accessible internalhydrogens).

associated with covalent binding of one-half a molar equiva-lent of 10-UDYA (Table I) also argues strongly for the pres-ence of two lauric acid hydroxylases, one that oxidizes 10-UDYA and is inactivated by it, and one that simply does notaccept 10-UDYA as a substrate. he two w-hydroxylase ctiv-ities could be due to two distinct isozymes or to post-transla-

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

tional modification of a single isozyme.The partition ratio for the inactivation of cytochrome P-

4 5 0 ~ ~ ~ ,efined as thenumber of moles of 10-UDYA oxidizedper mole of inactivated enzyme, is exceptionally low. If allow-ance is made for the fact that only roughly one-half of theenzyme can be inactivated, each molecule of the vulnerableenzyme appears to oxidize three molecules of 10-UDYA beforeit is inactivated, two of which appear as 1,ll-undecandioicacid and one of which is covalently bound to theprotein. Theinactivation reaction is not well coupled to utilization ofNADPH because the NADPH consumed in the short burstof activity during which enzyme inactivation occurs indicatesthat approximately 20 mol of NADPH areconsumed per moleof enzyme that is inactivated (Fig. 7). The enzyme thus turnsover approximately 10 times per molecule of 10-UDYA thatit oxidizes. Nine molecules of NADPH are presumably con-sumed in uncoupled reactions that reduce molecular oxygento hydrogen peroxide or water (Ref. 52 and references therein)and only one in the actual oxidation of 10-UDYA. The effi-ciency of the inactivation of cytochrome P-450 by agents thatalkylate the prosthetic heme group is substantially lower thanthat observed here. The partition ratios for substituted phen-ylacetylenes range from 38 for p-methylphenylacetylene to 4for p-nitrophenylacetylene (42), and the partition ratios forsimple olefins fall in the 100-300 range (28).The high efficiency of the inactivation reaction suggeststhat protein alkylation occurs before the ketene diffuses outof the active site. The increased polarity of the ketene metab-olite makes it likely that it has a weaker affinity for theenzyme than does lauric acid (K ,= 11 p ~ )r 10-UDYA (Ks= 14.6 p ~ ) .owever, the concentration of the ketene metab-olite if it diffuses quantitatively into the medium before italkylates the protein cannot exceed 1 p ~ .t is unlikely thatthe high specificity and efficiency can be reconciled withformation of the alkylating agent at concentrations substan-tially below those of the probable K, value.The active sites of the lauric acid w-hydroxylases must behighly structured in the vicinity of the activated oxygen tosuppress the highly favored w-1-hydroxylation (Table 11).This reaction control is probably exerted, as suggested byexperiments with model metalloporphyrin systems (161,bystructuring the active site so that only the terminal methylgroup reaches the activated oxygen. The tolerance of theenzyme for fatty acids of somewhat different chain lengthssuggests, furthermore, that the erminal methyl specificity isnot governed by specific interactions of the protein with thecarboxyl group. Access to the oxygen is therefore probably

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Lauric Acid w-HydroxylaseProtein Versus Heme Alkylation 18645controlled by steric constraints within the catalytic site, asillustrated schematically in Fig. 12.The structural features thatavor w-hydroxylation of lauricacid also favor w-oxidation of 10-UDYA. Since oxygen addi-tion to the erminal carbon is concerted with migration of theterminal hydrogen to the icinal carbon (26), the w-specificityof the enzyme leads to exclusive formation of the ketenemetabolite that probably acylates a nucleophile in the cata-lytic or substrate binding sites (Fig. 11). Conversely, the factthat the inactivation of cytochrome P-45b by phenylacety-lene is quantitatively accounted for by heme N-alkylationeven though it produces substantial amountsof phenylketenesuggests that cytochrome P-45b does not have a suitableactive site nucleophile (42).The active sites of the two lauric acid w-hydroxylases inour purified preparation differ markedly in that only one ofthe two enzymes is inactivatedby 10-UDYA.The observationtha t conversion of 10-UDYA to the diacid metabolite ceaseswhen the vulnerable enzyme is inactivated indicates that theresistant enzyme accepts lauric acid but not 10-UDYA as asubstrate. The extraordinary specificity of the resistant en-zyme for the saturated terminusof lauric acid thus protectsit from inactivation by 10-UDYA. Furthermore, neither iso-zyme appears to accept the terminal olefin as a substratebecause (a) oss of epoxidase activity caused by the olefin isnot accompanied by loss of lauric acid w-hydroxylase activity,and ( b ) nactivation of lauric acid w-hydroxylase by 0-UDYAoccurs very quickly but ts inactivation of the epoxidaseactivity occurs relatively slowly. Th e epoxidation thus appearsto be catalyzed by a trace contaminatingsozyme not involvedin the w-hydroxylationof lauric acid. The active sites of bothlauric acid w-hydroxylases thus exclude the terminal olefinanalogue and one of them also excludes the terminal acetyleneanalogue of lauric acid. A comparison of the active sites ofthese two enzymes should prove highly informative withrespect to the factors that control substrate specificity incytochrome P-450 catalysis.

Acknowledgments-We would like to thank Jennine Lunetta forexcellent technical assistance in enzyme purification, Barbara Swan-son and Mark Watanabe for obtaining the mass spectrometric data,and Dr. Angela Wilks for the preliminary SDS-PAGE/radiographyresults.REFERENCES

2. Tamhurini, P. P., Masson, H. A., Bains, S. K., Makowski, R. J., Morris,1. Kupfer, D. (1980) Pharmacol. Ther. 11 , 469-4963. Okita, R. T.,arkhill, L. K., Yasukochi, Y., Masters, B. S.S.,Theoharides,4. Needleman, P., Turk, J., Jakschik , B. A., Morrison, A. R., and Letlowith,5. Holm, K. A., and Kupfer, D. (1985) J . Biol. Chem. 260 , 2027-20306 . Williams, D. E., Hale, S. E., Okita, R. T., and M asters, B. S. S. (1984) J.7. Yamamoto, S., Kusunose, E., Ogita, K., Kaku, M., Ichihara, K., and

B., and Gibson, G. G. (1984) Eur. J. Biochem. 139 , 235-246A. D., and K upfer, D. (1981) J.Biol. Chem. 256 , 5961-5964J. B. (1986) A n m Reu. Biochem. 55 , 69-102

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Ogita, K., Kusu nose, E., Yamamoto, S., Ichihara, K., and Kusunose, M.Kaku, M., Ichihara, K., Kusunose, E., Ogita, K., Yamamoto, S., Yano, L,Shak, S., and Goldstein, I. M. (1985) J.Clin. Inuest.7 6 , 1218-1228Romano, M. C., Eckh ardt, R. D., Bender, P. E., Leonard, T. B., Strau h, K.Brash, A.R., Jack son , E. K., Saggese, C. A., Lawson, J. A., Oates, J. A.,Roberts, L. J., 11, Sweetman, B. J., and O ates, f A. (1981) J. Biol. Chem.Bains, S. K., Gardiner, S.M., Mannweiler, K., Gillett, D., and Gibson, G.Hardwick, J. P., Song, B.-J., Huberm an, E., and Gonzalez, F.J. (1987) J.Cook, B. R., R einert, T. J. , and Suslick, K. S. (1986) J . Am . Chem. SOC.Nappa, M. J., and Tolman, C. A. (1985) Inorg. Chern. 24,4711-4719Khenkin, A., Koifman, O., Sem eikin, A,, Shilov, A., and Shteinman, A.Fontecave, M., and M ansuy, D. (1984) Tetrahedron 40 , 4297-4311Fmmmer, U., Ullrich, V., Staudinger, H., and O rrenius, S. (1972) Biochim.Morohashi, K., Sadano, H., Okada, Y., and Omura, T. (1983) J. Biochem.Biophys. Acta. 28 0 , 487-494Terelius, Y., and Inge lma n-S und be~ ,M. (1986) Eur. J. Bioehem. 161,(Tokyo)93 , 413-419Vatsis, K. P ., Theoharides, A. D., Kupfer, D., and Coon, M. J. (1982) J.303-308Ortiz de Montellano, P. R. (1986) in Cytochrome P-450: Structure, Mech-Biol. Chem. 257,11221-11229anism, and Biochemistry (Ortiz de Montellano, P. R., ed) pp. 217-272,Plenum Publishing Corp., New YorkKerr, J. A. (1966) Chem. Reu. 66 , 465-500Ortiz de Montellano, P. R. (1985) in Bioactiuation of Foreign CompoundsOrtiz de Montellano, P. R., an d Reich, N.0. (1986) in Cytochrome P-450:(And ers, M. W., ed) pp. 121-155, Academic Press, New York

pp. 273-314, Plenum Publishing Corp., New YorkStructure, Mechanism, and Biochemistry (Ortiz de Montellano, P. R., ed)Ortiz de Montellano, P. R. (1988) in Progress in Drug Metabolism (Gibson,G. G., ed) Vof. 11 , Taylor & Francis, Basingstoke, England, in pressGan, L. S. L., Aceho, A. L., and Alworth, W. L. (1984) Biochemistry 2 3 ,3827-3836

(1983) Biochem. Int. 6 , 191-198and Kusunose, M. (1984)J.Biochem. (Tokyo)96,1883-1891M., and Newton, J. F. (1987) J . Biol. Chern. 26 2 , 1590-1595and Fitzgerald, G. A. (1983) J . Pharmacol. Ex Ther. 2 2 6 , 7 8 - 8 7256 , 8384-8393G. (1985) Biochem. Pharmacol. 34,3221-3229Biol. Chem. 262 , 801-810108,7281-7286(1985) Tetrahedron Lett. 26,4247-4248

Gan, L. S. L., Lu, J. Y. L., Hersh kow itz, D. M., and Alw orth, W. L. (1985)Ortiz de Montellano, P. R., and Reich, N. 0. (1984) J. Biol. C h e m 2 5 9 ,Biochem. Biophys. Res. Commun. 129 , 591-5964 l R f L " l A lCalacoh, C. A., and Ortiz de Montellano, P. R. (1986) Biochemistry 25,_ " _ _ _ "~ I ~ L A I ~CaJacoh, C . A., Shep hard, E. A., a nd Ortiz de Montellano , P. R. (1987)Chan, W. K., CaJacob, C. A., and Ortiz de Montellano, P. R. (1988)FASEB

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Macaulay, S. R. 11980) J. Org. Chm. 45 , 734-735Valicenti,A. J., Pusch, F. J., and Holman, R. T. 1985) Lipids 20 , 234-242Corey, E. J., and Suggs, J. W. (1975) Tetrahedron Lett. 2647-2650Fieser, L. F., a nd Fieser, M. (1967) Reagents for Organic Synthesis , Vol. 1,Fieser, L. F., and Goto, T. 1960) J. A m . Chem. SOC. 2,1693-1697Shephard, E. A., Pike, S. F., Rahin, B. R., and P hillips, I. R. (1983) Anal.Komives,E. A., and Ortiz de Montellano, P. R . (1987) J . Biol. Chem. 2 6 2 ,Waxman, D. J., and Walsh, C. (1982) J. Biol. Chem.257,10446-10457

Lowry, 0. H., R osebmugh, N. J., Far r, A. L., an d Randa ll, R. J. (1951) J.Laemmli, U. K. (1970) Nature 227 , 680-685Omura, T., and Sato, R. (1964) J. Bioi Chem. 239 , 2370-2378Gorsky, L. D., an d Coon, M. J. (1986) Drug Metab. Dispos. 1 4 , 8 9 - 9 5Phillips, I. R., Shephard, E. A., Bayney, R. M., Pike, S. F., Rabin, B. R.,Thomas, P. E., Reik, L. M., Ryan, D. E., and Levin, W. (1981) J. Bwl.Heath, R., and Carter, N. (1983) Bmhem. J. 2 1 2 , 5 5 - 6 4Towhin, H Staehelin, T., and Gordon, J. (1979) Proc. Natl. Acad. Sci. U.Chem. 256 , 1044-1052Hawkes, R., Niday, E., and Gordon, J. (1982) Anal. Biochern. 119 , 143-S. A . 76;'4350-43541A7

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18646 Lauric Acid ~ - H y d ~ o x y ~ erotein VersusHeme A l k ~ ~ t i o nS ~ P P L E h ~ E ~ T A R YA T E R I A L

t oT HE CAT AL YT I C S I T E OF RAT HE P AT I C L AURI C ACI D W HYDROXYL AS E

P r o t e i n vs Prosthet icem el k y l a ti o n i n t h e - Hy d r o x y l a t i o n fA c e t y l e n i ca t t yc i d sClaire A. Calacob.William K. Chan. ElizabethShephsrd, nd

Paul R. OrtizdeMontellanoM*uri.lr GlycerolGoldabei) , yanogen romide..8-diamin~lctanc. 10-

u n d e c m - l o l . n d . l l - u n d a c a m d i o i c a c id were fromAldrich: NADPH. DETAPAC.DBAE-Scphacel, CM -Sap hws e 68. CM-Scphadex C5O . Scpharose 4B. sodiumcholate.c l o f i t i c i d . and ctby1 lofibrate were from Sil lnu; dilavroylphosphalidyIcholine WIIf rom Serdlry ResearchLabora tories Pon Huron. MI): Bio-GelHTP ydroxyl;lpatite.Chsler res in , nnd horseradish eroxidase-conjugated goat ant i - rabbi t IgG were fromBie-R.d: 2:S"ADPScpharore 4 8 was from Pharmacia. Emulgcn was kindly rovidedby KAO At lu Corp. (Tokyo. Japan) . l -14Cllauric r i d (15.30 mCiImmol) and 14ClKCN(5 3mCiimmo1) were obtained romAmen ham. Unlabeled IO-UDYA was synthesizedas previws ly described 31) .Deionized. glass distilled water wa s used for al l biologicalwork.

radiolabeledO-undecynoic cidScheme I) . synthesis f -dccyn-1-01, was carricdout by themethod of Macaulay (3%. Sodiumhydnde (50% in 011 9.48 g. 0. 2 mol) wasplaced in a 5 0 0 mol two-neck romd bottom flzme.driod flask undcr nztrogen an d wasfreed of mineral oi l by washingwith exane. The olid was then uspended in 14 8 mlc m l e d in an ice bath. A solution of 3-dccyn-1-01 (3.9 g,25 mnol) in 79 ml of .3.of I . 3 d i ~ i ~ o ~ ~r d the mixturc was s t t r red .170C for 2 hr b c f w it wasdipminopropane was then dded ropwisewith a dropping unnel ndhe esultingmixture w u stirred at 55OC overnightand Was then e m l e d u) OC in an ic e bath. Aftcrconcentrated HCI was added ropwise to bring he pH IO approximately 3, themixturewas xt racted wi th ie thyl ther ndhe ombined ether extracts were washedremoval of the olvent at a rotary vaporator. an d distillation nder vacuum yieldedsequentially withwater and saturated queous NaCl. Drying over anhydrousMgSO4.2.89 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

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Lauric Acid w-Hydroxylase rotein VersusHeme Alkylation 18647employed (391. To a so l ~ t i on f the epoxide (0.43 g. 2 mmol l in 10 ml oftetrahydrofuran wa r added 0.8 ml of 30% aqueous perchlortcacid and the mixturewa s nllawed 10 stand for 6.5 hr ar room temperature under a nitrogen atmosphere.Water ( l o ml ) was then added an d the mixture was extracted thrice with 25 ml ofdiahyl ether. The combined organic layers were washed with water and satmatedNaCl solution and were drlcd over anhydrous sodium rulfatc. Solvent removal at arotary evaporator loliowcd by ~ ~ ~ ~ y ~ t ~ l l i ~ ~ t i ~ "rom I:[ hexane:ethyi acetate provided0 I g (0.4 mmol. 20%) of he 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): IR (nujol) 3374(hydroxyl) and 1694 cm.1 ( ca rboxy l ) .

1 1 , l L . D i h y d r o x ~ d o d P r a n o i c Acid. The procedure of Ficrer and Got0 Was

Animals. S p r a g u e ~ D a u , l e ymal e rats (180-200 g) were induced with clofibrate intwo waya. In early expermeno, rats wexe fed wrth Normal Piolein Test Rat Diet (ICNBiochcm!cals, Cleveland) supplemented with 0.4% cloiibric acid for 14 days. In laterexperiments. the ra n were inpcted intraperitoneallywith rhyl lofibrate at a dose of400 mglKg once 3 day fo r three days. The animals were used to prepare th e enzyme24 hr after the last dose of ethyl lofibrate. There were no noticeable differences iothe e n z y m e obtained from the m s nduced by the two procedures.

Shephard et 21. (40). Cytochrome h5 and cytochrome P450b were purified from theEnzymes . CytochromeP450 reductase was prepared by the procedure ofl ivers of phenabarbital-pretreated rats by the procedure of Waxman nd Walsh (41).The ~ m p l e m e n l a t i o n of there prcparotmnr and the quality of the proteins obtained iothts l abor ator y have heen reported (42). Lauric acid w-hydroxylase wa r purified fromderfiibed by Tamhurini et al. (2). From 20@0 mg of microsomes (20 rats) 10-50 "molthe I ~YPISf clofibrate-induced Sprdgue-Dziwley male rats (180 200 g) essenual ly aso i purif ied enzyme (approximately 1% yield) wa s obtained. The punty of the enzymesw x assessed by SDS-PAGE In a 7.5% gel by the procedure of Laemmli 143).

bovine .;erum albumin as ?he rtandard 144). The lauric acid o-hydroxylase ac t iv i t yArrays. Prolein ~oncent rat i on3 were measured by the method of L o w r y with

radiolabeled lauric acid a f m their separation by HPLC BI described earlier (31).wa s mcarured by ]>quid scmti11at~on counting of the metabolites produced fromCytochrome P.450 was quantitated hy dtfference apecrrorcopy of the reduced. CO-saturated versus reduced enzyme on an ArniiicoDW-2a instrument. The 450-490 nmrhrorbmce difference and an at inct$on coefficient of 91,000 M- I Em-1 w e r eemployed far Lhls purpose (45). To determme the abiolute position of the Soret banduf P45OLAo. ytochromeP-450 (0.85 nmoliml) in IOU mM potassium phosphatebuffer. pH 7.4. containing 20% glycerol and 251) uM DETAPAC wa s reduced withdilhronite and the ramplc was split into two ~uvettes. After the baielinc was recordedf r om 400 10 500 nm at 0.2 nmirec. carbon monoxlde was bubbled into the sample celland ih e difference s i l e c t ~ u mw3a recorded.

hydroxylase wa s reconstitutedwtth ytochrome P450 reductase and. except whereindicated. with cytochrome h5 by the gtnersl procedure of Gorsky and Coon (461. Theconccnlr3rmns given ~n thmi and the subsequent procedures are rhose in the f ina lincubelton mixtures.Typically.ufficicnt 100 mM potassium phosphate (ptl 7 . 4 )cytochromeP450 ( 0 . 5 nmoliml). cytochiame P450 reductase ( I nmoliml), andhuffercontaining 20% glyccrol and 25 0 aM DETAPAC wan added to a mixture ofd i l3u r oy lphospha t ~dy l - ~h~ l~" ~40 ~ g l m l ) 10 make the final incubation volume 0.45nil. This mjxture war Incubated for 5 mi" a1 25C before adding cytochrome b5 (1nmaltml). After an add%tzand5 mi n at 25C. 5 ul of dimcihylsulfooxide or a similarv0Iumc of d~melhyl iul fox~deontalnmg the substrate or inactivat~ng g e n t wa s added.incubated for 1 ~ 5 in inactivatmn experiments) or 6 mi" (activity assays) at 2 S T . InNADPH (0.5 m h l l was then added to iniliate [he reaction and thc mixiwe wasthc inactivation expeamentr. a 100 pl aliquot of the tncubalion mixture was thentransferred to a prewarmed mlx iure o i d ~ la u r o y lp h o s p h a ~ id y r c h o i i n eI? pi ml ) .7.31 buffer conraining 20% glycerol End 25 0 uM DETAPAC ( f i n d voliime 1.0 mi). TheNADPH (0.5 mM). and 14C~Iauricacid (0.55 mM ) n 100 mM potassium phorphote (pH

otheiwisendicated) efore the reactmn was quenched by adding 10% ("1") aqueousmixture wds m u b a t e d :it Z S T for the mdicatedperiod of time (6 mi" unlesss u l l u r ~ od . Lauric acid and XIS met abol ~t erwere then extracted and analyzed by highpresrure Isquid ~ h ~ ~ m a t o ~ ~ a p h ys reporled previously (31). Incubation o i theenzyme for various t ime perlodr in the bsence of substrater or inhibitors ollowcd bydilution and a s a y as described hcre showed [hac the activity assay employed ininhihttor studics IF Isnear

EnzymeReconsti tution nd lncubalian Conditions. ariiied lauric acid 0-

cytochrome Pa50 reductase 11 nmollml), Sl lauroy l -phosphar idy l~ h~ l ine4 0 pgiml) .and inhibitor (0.3 mM1 ~n dimethylsulfox~dcor 0.55 mM lauricacid in the samevolumf of dimethyl,ulforide were Incubated for 5 min at 2 5 T beforeadding sufficient100 mM potassium phosphate (p H 7.41 buffer containing 20% glycerol and 250 pMDETAPAC s o that the finalmubarion volume would bc 0.5 ml. Cytochrome hg (Inmolimi) was then added and th e mixture was incubated for r further 5 mi n at 25Cbefore NADPH (0.3 mM ) wa s added to initiate cacalylic tumover. The absorbance at340 nm was followedcontinuo~sly for I S min on a Hewlett Packard 8450Adiodearray IJViVIS spectrophotometer against a reference cuvctlc that containedd ~ l u u t o y l p h o s p h a u d ~ l ~ h ~ l i ~ =40 pglml). 0.3 m M dimethylsulfoxide, 20% glycerol, an dchanges were converted to nmolcs usmg the cxtinclion c o e f f i c ~ e n t or NADPH of 6.22 x250 uM DETAPAC an 100 mM polasslum phosphale (pH 7.4) buffer. The absorbance10 3 M.1 cnt-1.

NADPH Consumption Experiments. Lauric acid .hydroxylase ( 0 .5 nmaliml),

Cytochrome P450. A standard I ml mcubarion war set up byncubating cytochromeP450 (0.2 nmolimll. one of the following ~oncent~alionsf cytochrome P450 reducurc(0 . 0.1 , 0.2, 0.4. 0.7. 1.2. 2.0 nmollml). and dilauroylphosphatidylcholine (40 glml)fo r 10 min at ZST. To ihts was added the requisite amount of 10 0 m M potassium

Catalytic Dependence on Rat io of Cytochrome P450 Reductaseo

phosphate (pH 7.4) buffer containing 20% glycerol and 250 uM DETAPAC.14C-Sodlun1laurate (0.55 mM) in dimethylsulioox~de.and NADPH 12.0 mMi. After nrubaling for 5min at 2SC, the reactions were quenched by adding 10% sulfurlc m d 2n d theproducts were analyzed as usu3I.

male New Zealand While rabbits using the protocol reported by Phi l l~pr t 81. ( 4 7 ) .Anlibodier. Antibodies 10 purifted lauric acid w.hydroxylase were raised in twoEach rabbit was injected in the subrcvpular space with 5 0 ug of he antigen mtxed l : t( v i " ) with Freund's adjuvant. Three weeks later. the rabbits were ~nj ect ed wlth 25 u gof he antigen similarlymired will>Freund's adjuvant. Five weeks later. the rabhitswere given a 12.5 ug booster injection of the antigen and 7 drys la ter blood w mwithdrawn from the marginal ear Yein and was allowed lo clot at room rempei:llurefor 30 min. The c l o t w3s broken. stored at 20C overnight, and centrifuged at 10000 6for I S mi n ar 4C. The antiserum was decanted and stored in aliyuolr a1 -20C. Theboorter and antis erum ollection steps were repeated ai two month m t e r v ~ l s Thepresence of antibodies specif ic lo lauricacido-hydroxylase ua s conflrmcd hyOuchterlony ouble-immenodlffuiion analysis as described by Thomas ct dl . 14x1. Thcantihodier were found 10 react wt h the o.hydrorylase hu t not ulrh P 4 5 0 h o r P 45 lkhy Western blot analysis.

uninducsd and induced rat h v e r microsomes was carried out by the procedureWestern blot analysis of the con~entrat ions of lauric acid o.hydroxylnsr inprovided with Bio~Rad its based on thar u i Towbin et 91. (49). Sample? ofpuziiirdI m n c acid o.hydroxylase rangmg from 3-10 pmol. mmosomCsrom umnduced fa tlivers (E0 and 40 p g total protein). and microbomcs from clofthrate-mduced i i t l h e rtransferred to nitr~cellul~sen a B w R a d T ~ a n s bl o rapparatus operated at 1011 mAI15 and 7.5

ug total protem). were run together on 7 % SD S gels The proteins wereovermght followed by 200 mA far I hr.Non-rpcciiic protein hinding SNCE %ereblocked with gelatin. Goat anta-rabbu IgG con~ugaledw t h horseradish pero.vdase wasemployed as the second anrihody and 4-chloro-1-naphthol was used as the pcioridvsesubstrate (50 ) . The protems stained by this procedure wcic quant mt ed bydensilometry. I h e concenrratiun~ f lauric acid -hydroxylase were derermmed wirhrespect to the linear sectmns of Ihr atrndard c ur ve s gentrated wlth authentic o-hydroxylase.

10-Undecynoie .4eid Metabolitcs. ytochrome450LAco ( I nmoliml).dilruroylphoaphat~dylcholine (40 ug/ml ) were incubated 5 mi n 81 2 5 T and wfre thencytochrome P450 reductase (2 nmol/ml). 11-14CCIUDYA (0.6 mM1. andtaken up in 100 mM potassium phosphate (pH 7 4 ) buffer containing 2 0 1 glycerol and250 pM DET, 'AC. Cytochrome bg 12 nmollmll wa s added (f inal vOlUmC 2 m l) and themixture incubated 5 mi" ai 25C before NADPH (1 mM ) BI added to initiate ther e ~ c t ~ o n .fter 5 mln 81 25C. the mixture wa s quenched wll h 0 8 ml of 10 % HZSOIThe mixture was extracted with diethyl ether 13 L 4 ml) and the combtned ex t r i m \and unlabeled IO-UDYA

(2 pwol) and l . l t-underanedio~caced (2 pmd) were rddcd.were passed through a r m d l pad of riltca gel m a disposable p&pe!te.concenlratcd 10 av o l ~ m eof 2 ml under a stream of nitrogen. combined wrth2 ml of ethercrldiazomethane, and allowed to stand for 45 mi" a1 room temperature. The s ~ l v e n tw;nthen removed under a stream of nitrogen rn a hood and was replaced with SOU pl of1: l ~c~tonitrllc:waler.A 100 pl alquot oi the resullmg solution wa s analyrcd by hlghpressure l i q u i d ~ h , - ~ t * g ~ ~ ~ h yn a I 5 x 4.6 cm Dupont Zorhax ODS 5 um ClX revcrscphase 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 va i mbl e wavelength detector.

reconstituted ytochrome P450 LAw as desfrlbed above for IO4JDYA. l n t c r n dstandards (12-oxod odecan oic acrd, 11.12.epoxydodecsnoic acid, 1.2ektracted twice withdlelh yl rrher. 'The erfiacts were faltered through rma!l ulic'' geldihydroxydodecanoic acid) were then addcd un d the lncubat~onmixtures werecolumns beiore iazomethrne ( 3 ml ) was added to each vial and the v i a l ? were c.gppedand allowed 10 sand 45 ml n a: room temperature. The iolvent and exces sdzaromethane were then remowd under 3 i l r e e m of nmosen and 0 .5 m i of 31: lYmethanol.watcr was added to each vial. The resulting SOIUIIOIIEwere analyzed by I IPL Cusing the s y ~ t e ~ ld conditions used for the analysis of lauricacid tneraholito.

Dodrcenoie A c i d Metabolites. [ I-14CII -dodeccnoir cid wa s mcuhotcd with

two dlfferent methods. onc involving f i l t rat ion throughWhstman filler paper and lh eProtein Labelingwith 11 -14CIUDYA. These exper!menrr w e r e carried out byothcr column chromatography on Sephadcr G-25. The incubation pioceduie. whrchcytochrome P450 reductase (2 nmollml). I I -14CIUDYA ( 0 6 mM) andwas the same m both cases. involved Incubation of cytochrome P450LAw (I nmnllml).d i h u r o y l p h o s p h a l i d y l ~ h ~ l i n e40 pgiml) fo r 5 mln at 2S'C prior 10 adding 100 mMpotassium phosphate (pH 7.4) b uffer containing 20% glycerol and 25 0 uM DETAPAC.Cytochrome bg (2 nmol lml ) wa s then added and thc mixture wa s incubated a further5 mi" at 25C before NADPH (1.0 mM) was added 10 mitivie the reacuon. Afier 3 nlmadded and thc mixture was allowed 10 sand for 5 m m The precipitated prolem wa rat 25C. the incu bation wa s put on ice and 4

ml of cold 25% trcchloroacet~actd WAScollectedby Suction filtration i h rwgh 2.4 cm diameter Whatman GR B f?lrcr disc). Thedscr were rmsed cold 25% trichloroacetic acid (4 K 1.5 ml). old Rh tr~chlnroaccuc rc d(8 x 1.5 mil. and acetone (4 x 1. 5 mi). Conrrvl experimcnrr indicated t h ~ t he laheledhpid was not retained y the f i l te r s after thir procedure.

mi". The incubation mixture wa r then put on ice until i t could k loaded on a 1 I 35I n the alternative procedure. the clrmplete reaction mixture was incubated for 5

cm column of Sephadcx G-25 fine grade) that had been swollen an d preequihbrated0.5% (w / v ) sodium cholate. The column was eluted with the Same buffer system an d 7with 50 mM Triris-acetate buffer (pH 7.41 containing 20% glycerol, 0.1 mM EDTA. andmi" fractions werr collected.The absorbance of each fraction was measured at 412 nmto determine the P450 cantent and the sample was subjected to liqutd scintil1monc o u n n n g .

In some instances. 5 min fractions were collected from the Stphadex G-25 columnand those fractions containing the hemoprotein were pooled. A 25 pl aliquot wits takenfor SDS-PAGE and the rest war loaded onto a 1 x 3 cm (2 ml volume) column of 2 ' scontaining 20% glycerol. 0.1 mM EDTA, and 0.5% sodium cholate. The cytochrome P450ADP~Scpharore4 8 that had been prcequilibrated with 50 mM Trir-acetate p H 7.4and hs were eiurcd with the same lris buffer containing, in addition, 0.75 M KC]. heKC1 u.31 added to minimme ionic i nler hcli on~ between the hemoprote m andflavoprotein. The reductase was then eluted wish the same T m bufieer containing 5mM N A D P + . A 30 pl sample of each fraction w85 rcmovcd for SDS-PAGE and heremainder wa s subjected to laquid sciotillation counting.

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18648 Lauric Acid w-Hydroxylase Protein Versus Heme AlkylationP4 5 f l L Aw ( 1 .3 3 nmollml). cytochrome P450 reductase (2.66 nmollml). I O - U D Y A (0.6Spec troscop icChangerCaused byC o v a le n t Labe l ing . ys tem on ta in ingm M ) . dllluroylphorphatldylcholinc (40 pg lm l ) . 20 %glycerolan d 25 0 pM D E T A P A C nI00 mM po tasuum hosphr le u f fe r pH 7.4) wa s reconr t iwted as usual (10131 volume0.75 ml) . N A D P H ( 1 . 0 m M ) as then added and the mi xture ncubate d 5 mi " at 25 'Cdercrtbed. The nzymc con taming fractions were poa lcd and concentrated withbefore i t W B ~ ooled m ~ c e nd put through

a Sephadex G-25 column as alreadyCcntricon fsltcrs (10.000 MW-cutoff). Similar xperiments were carried out w l th a u tN AD PH .wi thou t O-UDYA.an dwithou tbo th O-UDYA and NADPH.The absorbance at412 n m o f il 0.5 ml volume of each sample was adjusted wtth Tr i rbu f fe run t i l al l thev d u c s (c.g. the protein conccntratmnr) were the same The ample blained from theincubation wkthaut bo th O-UD YA and NA DP H was then a lanced against i t r c l f rom35 0 lo 500 nm on the Hewlctt ackard mde rray pectrophotomerer nd theabsorbance difference spectrum rccorded. he ample cuvel te was theneplaced inlurn by each of th colhcr lhrce sampler ( - N AD PH .+ IO - U D YA:+NADPH. IO-UDYA: and+ N A D P H .+IO -UD YA ) and the absorbance difference spectrum ecorded.

R E S U L T S

P~SOLA,. the cytochro meP450 thdt catalyzes w-hydroxy la tmn of lauric actd. wa spurlfted from the livers of c la f~h ra rc -p rc l rea tcd ats by the procedure f amburm i etill. 12). The nzyme thus ohtamed had a spcclfic content that ranged f rom 7 to 12 nmoll '450lmg rotein. he peetftc conlcnt repor ted y amburml et 31. is 12.8 nmolP4501mg protetn ( 2 ) and byHardwnck et al. 16-18 nmol P450lmg protem ( I S ) . SDS-PAG E of the enzyme Figure I ) shows that our enzyme preparation Contains twoa l m o s t unresolved bands that ru n more s lowlyhan y toch romeP450b . In slightly lesswell purified reparations a well resolved lower bands bserved (e+. Figure 9 ) . Theprotein esponsible fo r th in lower band ts not detectablynvo lved in lauric acidhydroxy la tmn because the same resulls are ohtamed in i ts resence or absence. Th eabsorbance maximum of the reduccd. carbon mononde.haund nzyme. as repor tcd yTamburini et P IS at 45 2 nm (2).

P u r i f i c a t i o n n d h a r a c t e r i z a t i o n of L a u r i c c i d - H y d r o x y l a s e .

abscnce of cytochrome b5 wa s 8 - 1 1 n m o l l n m o l P4501mm. I n the presence ofThe production of I I - and 12-hydro ry l ru r ie acids by the nzyme at 37'C in thecy loch romc bg . the catalytic ctiv ity wa s 18-20 nmollnmol P450 lmin at 37C. Therevalues were obtained at a 2 : l a t io of cytochrome P450 reductase to cytochromeP450because thiss the mm imum ratto requ ired o rmax imum c t iw ly (no1 shown).Tambur in i et al. report an aetlv ity of 43.0 nmol produc t lnmo lP450 lminn theabscnce of cy toch rome g ndHardwick et PI. a value o f 18.5 nmollnmol P4501min inthe presence o f ytochro me b5 ( I S ) . The 2-hydroxylatedaur ic c ld in the presenceof cytochrome 5 a c c o ~ n t s or 95.99% o f the p roduc t . I l -Hydroxy lau r icac id s usual lyP minor. ften arely etectable.metabolite. he roportions of the two products inpa ra l le lncuba t ionswi th ndwi thou t y toch rome gnd ica teha t y toch rome gstimulates fo rmat ion f thc 12-hydroxylated roduct y a factor of 2 IO 3 but has nomeasurable ffect on produe lmn f the I l - h y d r o x y la te d r o d u c t . a m b u r i n i e t alreportedha their reparmon gave at best B 6 : l a t io of the 12 - o l -hyd ro ry la tcdproducts (2) . Evidence that the 0 -1 hydroxy la t ions r imar i ly a ta lyzed y acontaminatingsozyme in the o .hyd roxy lsse repara t ions rovided y theobservationha thew-hydroxylase cliv ity eclinesmuch faster on storage than thc0 - 1 hydroxylase ctiv ity. wo ifferent nzyme repnralmn s tored at -79C for 1 Randmonths.espectively.etamed 15.20% of their 0- 1 hydroxylase ctiv ity buton ly pp rox ima te ly 0.1% of them o -hydroxy lase c t iv i ty . Well aged preparauonrconsequently xidizeauric cid lowly to a 1:6 mixt ure f the ro-and w - l products.

raised 10 pur i f ied ymchromcP450LAw in rabhilsecogmzcd the two poorlycsolvedhands of the purlfied nzyme reparation figure 21 but d id o t crossreact w i t hcytochromeP45flb or P450e. Ad d ln g ~nereasmg amounts o f the anlibody totncubattonr ofauric cldw it h thc r ec on rt rt ut ed y to ch ro me P 4 5 0 ~ ~ ~y r l e m r c v e d % .as cxpected. a concen tra t ion -dcpcnden tnh ih i l ion of t h e w - h y d r o x y la t i o n reactlo"(Rgure 3). The 0- 1 hydroxy la t ion IS also inhibited but not wtlh !he same conccntrmnndependence (now that the ac t ud magnitude of the w - l hydroxy la tmn 8s much lowcrthan that for the o-hydroxylation).The n t ibody at a mncen lra tmn of 25 mp. IgGInmolP 4 5 0 depresses the w-hydrox ylase cllv ity y a lmos t 0%.

l m m u n o q u a n t i t a t i a n of H e p a l i ca u r i c c i dy d r o x y l a s e .n t i b o d i c r

and

\

mo IgG I mmol e 5 0Figure 3. lnhlhiti on f auric acrd w - and 0 - 1 hydroxylation bym l l h o d l c r to

conditmn s n the presence of increnbing amounts of Ig G from cy toch romeP4 5 0 L Aw -cy toch rome 45 f lLAo . he yd roxy lase c I Iv i les were mcacurcd nder th e r tm d d r dimmunized rabbits. The experimental etails arc given tn Malcrlaln an dMcthodr.f rom uninduced and cloftbrate-induced rats. bared on a standard curve ertabllrhed

Western blot analyslr of the content of immunodetectableP45flLAw tn mlcro\ome%wlthncreas ing amounts of the authemic nzyme (Figure 2). establishhat ,he protun1s Induced several fo ld y lo f lb r r le . hemmunochemlca lmerhodndicale? thatP 4 5 0 L A W i s present in uninduced and inducedmtcrosomes at COnCenlrationS of IX-33cytochrome 450 in the Same microsomes y tffcrence pcctroscopy gtver v a l ue s forand 322-605 pmollmg protein.espectively. nalysis of the lo111 amount ofthe uninducedndmdueedmicrosomesf 1.34 and 2.03 nmallmg. rerpecllvely.These values indlcateha t y tochrome P45 0LA o represents only 1.3-2 5% of !he lmnlP450 content o f nmduccdmicrosomes nd 16.30% of t he enzyme i n lafihrate-Induced m m o s o m e s . Th is R- fo ldnduc t ion at the protem l c v e l agrecr WCII wuh thc17-foldnduction nn the o -hyd roxy lase c t iv i t ies of the same mmosomes. Th e 0 -1hydroxylase cttv ity. as expected tf it sp r ima r i ly ca talyzed by a differentsozyme. isonly levated .5-fold y clofibrate trealment.

The II- nd 2 -hydroxy la t ion f lauric acld yeconstituted cytochromes P450h andR e g io 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 o ny P150b and P ~ S O L A ~ .

P 45 0L A o has been quantitatively measured. As hown tn Ftgurc 4. P45flb gwer rhcI . and 2-hydroxylatedmetabo l l tcs in a ra t io f pp rox ima te ly : I . he rnmrryaelecriv ilyorhe process. defin ed a s (12 -hydroxy lau ra1e l l -hyd ro ry lau ra te ) x(number of I I -hyd rogenr) / (number of 12-hydrogens). ls 0.083. In ConIrast. P4 5 f l L Awgiverhe I I - and 2-hydroxylated metabohter ~n approximately a 1:17 ratlo. Thcprimary electiv ityor the reaction atalyzed y 450LAwshcre lo re pp rox tmatc ly11.3. The two enzymes i f fe r n he i r nmary e lec t iv i ty y a factor o f about 136

P450b p450LA w

cy toch romes P450b an dP450LAo.The o l id bars on the left are the values fo r 1 1 -hydroxy lation nd the crosshatched bars on the right the values far 12-hydroxylation

Figure 4. Com parison of the formation of 1 1 - and 2-hydroxylauric cids y

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Lauric Acid ~ - H y ~ r o ~ y l ~ erotein VersusHeme A l ~ l a t i ~ nThe specificacttvity denoted by each bar is given above it I n units of nmolproducl lnmol enzyme.different. Under our incubation conditions. the rates of formation of ihc 11 - and 1 2 ~The ra m of oxidanon of lauric acid by P450b and P 4 5 0 L A m are also qrltehydroxylauric actds by cytochrome P45ob were 0.48 and 0.06 nmollnmul P450imln.rcspectively.The orresponding valuer for P 4 5 0 ~ ~ 0ere 0 . 8 4 and 19.21 nmollnmolP450i mm. respectively. The net rate of oxidation ai lauricacid wa s therefoore 0.24nmol lnmol lmm b y P 4 j o b and 20.05 nmollnrnollmln by P 4 5 0 ~ ~ o

Inactivated P $ S O L A o .N o ;onsumption of N A D P H is detected bymonitoring theN A D P H Canrumpl ion by Cytochrome P41iOl.A" and artially

absorbance at 340 nm in incubations of NADPH withoutauric acid. cytwhromedetected when cytochrome P450 reductase 1s present an d is ~ncrsmenral lyenhancedP ~ S O L A ~ ,ytochrome P 4 5 0 reductase, and cylochrome hs. NADPH consumpllon lsby the sequentml addition of cytochrome P 4 5 0 L A o and cytochrome b j (no1 shown).N A D P H consumption is doubled If the enzyme concentrations are doubled. Thus. 81 25oC. the rates of N A D P H consumption are 4 .34 and 6.77 nmollmin, respe;rwely, wtlh0. 5 and 1.0 nmollml of cytochrome P450. Addition of lauric acid 10 the completesys:em markedly increases N A D P H consumption. The itnear rate observed over thefirst 300 ~ e c dicatcs that N A D P H i s consumed at a tale of 1 nmoiimininmul P 4 1 0(Figure 7). Product formallon in (he same period is S nmolimininmol P4S0 in theabsence of cytochrome 5 and 10 " m ~ I l ~ i ~ i ~ m " 145 0 in it s presence.Approximrtely I nmol of N A D P H is therefore consumed per nmol of product in thepresence of cytochrome hg whereas in it s absence the consumprion of NADPH i s~ p p ~ ~ ~ i m ~ t e l ynmollnmol of product The primary cifect of cytochrome bs IStherefore 10 m r e a s e the efficlency of the reaction rather than LO increme the rate ofoxygen activation.

added 10 lhe incubation mixture. The change caused by IO-UDYA OCCUTS within theIn ~ o n l ~ a s t .A D P H consumption i s only lrghtly cimulrted when 1O.UDYA isf m t 100 seconds. after whtch N A D P H consumption i s roughly the same w t h andwithout IO-UDYA. The rapid hut small increment 10 NADPH con5umpson caused byIO-UDYA approxima!ely doubles if the enzyme concentrat~m s doublcd. The burst20 "mol of N A D P H per nmd of cytochrome P450. n view of the fact that$"crease in N A D P H consumption caused by addltton of I O ~ U D Y A S a p p r o x l m ~ t e l yapptortmately 2 molecules of 1 0 - U D Y A are oxidized per imctivation event (seebelow). one of whichhinds ovnlcntly to rhe enzyme. i t appears that 10 nmol ofN A D P H and the activation of oxygen are therefore much less efficicntly coupled 10 theNADPH are consumed per molecule of IO-UDYA t h u is oxidized. The con?umption ofox1dmon of IO-UDYA than lauric acid. Thc fale of the oxygen m the c a t r l y t ~ c ycle5that do out result in hydroxylation of IO-UDYA IS not known. If oxygen is reduced $0the l eve l of hydrogen peroxide. the ~nzyrne urns ov m nine T ~ C J efore it succeeds inoxidizin g rhe acetylenic substrate. If oxygen IS reduced 10 water. the enzyme goesthrough half as many futde catalytic cycles. !n contrast. there are essentially no fuulecacalpc cycles when lauricacid is nxidzzed I" the presence of cytochromehs.

dependent loss of lauric cid ydroxylase ctivlly or chromophore loss 1s detectedwhen reconstituted ytochrome P 4 5 o L A o is incubared wtlh 1-dcdecenoii acld andNADPH (not shown). Radiolabeled 11-dodecenoicacid is , nevertheless. eniymati~allyoxidized to a 1 0 : I mix tur e f 11,12-epoxydodecanoic acid and 12-oxododecano1c(Figure IO). Control experiments suggest that the diol arises bynmplc chemwalhydrolysis of the spoxrde during the incubation and isolation procedures but thenpproximately S mi" of incubation even thoughatlricacidhydrarylatton i s notaldehyde appears to be an enzymaric product.Epoxidat*on levels off afteratteneatcd. Preincubation of the reconstituted enzyme with IO-UDYA ICSUI~Sn tm edependent inactivation of the cpoxidation eact~on. but inaetivat~on IS not completeafter IO mi" whenasmacrivation oi lauricaci d o -hydro xy l ~ i on y IO-1JDYA ise~sen tialiy complete after I mi n (not shown).

Interact ion o f Cytochrome P 4 5 0 ~ ~ ~ithf-Dadecenoic rid. KO lime

"07o 1 0 2 0 30

Time (min)

metabolites of 11-dalecsnaicacid DDEA) as a function of the timc of incubationof the rad iolabeled lefinwith the reconstituted ytoehromc Pd XJL Ao system.l l , l Z - ~ ~ ~ ~ y d a l e c = ~ ~ i ~cid (l).2-onododccanoic cid ( e ) and il.12-dihydroxy-dodecanoicacid ( 0 ) .

Figure 10. Famation of the Il,l2-eporide. 12-07.0. and 11,12di ol

cytochrome P4S0 reductarc. and dilauroyl~phorphrl idylchol lne a) in the a h x n c e ofadded ruhrtrates, (b) i n thc presence of IO-UDYA. and IC ] an the pceseoce of lauricacld. N A D P H conrump..m isgiven PS the change I" the rhrorbance of the cofmor 3%340 nm. The experimental procedures are glv e n ~n Methods and Mater~ a lr .

Figure 7. Consumption of N A D P H by 9 system composed of cyrochromt P 4 5 0 L ~ ~ .

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