Proc. Nati. Acad. Sci. USAVol. 89, pp. 10507-10511, November 1992Biochemistry

Analogs of palmitoyl-CoA that are substrates formyristoyl-CoA:protein N-myristoyltransferase

(protein N-myristoylation/peptide N-tetradecanoyltranferase/substrateSaccharomyces cerevwsiae)

spefldty/molecular recognition/fatty acid auxotrophs of

DAVID A. RUDNICK*, TIANBAO Lut, EMILY JACKSON-MACHELSKI*, JEANETrE C. HERNANDEZt, Qi Lit,GEORGE W. GOKEL*t, AND JEFFREY I. GORDON**Department of Molecular Biology and Pharmacology, Washington University School of Medicine, St. Louis, MO 63110; and tDepartment of Chemistry,University of Miami, Coral Gables, FL 33124

Communicated by David M. Kipnis, June 29, 1992

ABSTRACT Saccharomycescerevasiae myristoyl-CoA:pro-tein N-myristoyltransferase (Nmtlp; EC 2.3.1.97) is an essen-tial enzyme that is highly selective for myristoyl-CoA in vivo. Itis unclear why myristate (C14:0), a rare cellular fatty acid, hasbeen selected for this covalent protein modification over moreabundant fatty acids such as palmitate (C16:0), nor is it obvioushow the enzyme's acyl-CoA binding site is able to discriminatebetween these two fatty acids. Introduction ofa cis double bondbetween C5 and C6 of palmitate [(Z)-5-hexadecenoic acid] ora triple bond between C4 and CS or C6 and C7 (Y4- andY6-hexadecynoic acids) yields compounds that, when con-verted to their CoA derivatives, approach the activity ofmyristoyl-CoA as Nmtlp substrates in vitro. Kinetic studies of42 C12-C18 fatty acids containing triple bonds, para-phenylene, or a 2,5-furyl group, as well as cis and trans doublebonds, suggest that the geometry of the enzyme's acyl-CoAbinding site requires that the acyl chain of active substratesassume a bent conformation in the vicinity of C5. Moreover,the distance between C1 and the bend appears to be a criticaldeterminant for optimal positioning of the acyl-CoA in thisbinding site so that peptide substrates can subsequently bind inthe sequential ordered bi-bi reaction mechanism. Identificationof active, conformationally restricted analogs of palmitateoffers an opportunity to "convert" wild-type or mutant Nmtsto palmitoyltransferases so that they can deliver these C16 fattyacids to critical N-myristoylproteins in vivo. nmtl81p containsa Gly-451 -* Asp mutation, which causes a marked reductionin the enzyme's affimty for myristoyl-CoA. Strains of S.cerevisiae containing nmtl-181 exhibit temperature-senstivemyristic acid auxotrophy: their complete growth arrest at 37Cis relieved when the medium is supplemented with 500 FMC14:0 but not with C16:0. The CoA derivatives of (Z)-5-hexadecenoic and Y6-hexadecynoic acids are as active sub-strates for the mutant enzyme as myristoyl-CoA at 24C.However, unlike C16:0, they produce growth arrest ofnmtl8lp-producing cells at this "permissive" temperature,suggesting that these C16 fatty acids do not allow expression ofthe biological functions of essential S. cerevisiae N-myristoyl-proteins.

Myristoyl-CoA:protein N-myristoyltransferase (Nmt; pep-tide N-tetradecanoyltransferase, EC 2.3.1.97) catalyzes thecotranslational transfer of myristate from CoA to the amino-terminal glycine residue of a variety of eukaryotic cellularproteins. These include a number of protein kinases, phos-phoprotein phosphatases, components of signal transductionpathways, and polypeptides involved in protein and vesiculartrafficking. In addition, both enveloped and nonenvelopedviruses encode proteins that are substrates for cellular Nmt.

Addition of myristate is required for full expression of thebiological functions of many of these proteins (1, 2).Nmt is encoded by single-copy genes in Saccharomyces

cerevisiae, Candida albicans, and Homo sapiens (3-6). Mostof the information about Nmt's kinetic mechanism and sub-strate specificities comes from an analysis ofthe S. cerevisiaeenzyme (Nmtlp). Nmtlp has an ordered sequential bi-bireaction mechanism (7):

myristoyl-CoA peptide4,

catalysis

Nmtlp -- Nmtlp-myristoyl-CoA -. myristoyl-CoA-Nmtlp-peptide -.myristoylpeptide-Nmtlp-CoA -* Nmtlp-myristoylpeptide -. Nmtlp

4, ICoA myristoylpeptide

The concentrations of myristic acid and myristoyl-CoA are atleast 5-fold lower than palmitate and palmitoyl-CoA in eu-karyotic cells (8-10). Nonetheless, Nmtlp appears to behighly selective for myristoylCoA in vivo (11). It is not clearwhy myristate is used for this protein modification ratherthan other, more abundant cellular fatty acids such as pal-mitate.The product inhibition studies used to define Nmtlp's

kinetic mechanism revealed that Nmtlp has functionallydistinguishable acyl-CoA and peptide binding sites (7). Thephysicochemical properties of the acyl-CoA presented toNmtlp can profoundly affect the enzyme's subsequent in-teractions with peptide substrates through cooperative inter-actions between the binding sites, consistent with the orderedbi-bi reaction mechanism (7, 11-13). Over 200 fatty acidanalogs have been tested in an in vitro assay of enzymeactivity to define the structural features of the acyl chain ofmyristoyl-CoA that are recognized by Nmtlp (11-13). Thesesurveys suggested that (i) the acyl-CoA binding site candetect the distance from the carboxyl to the co terminus of abound fatty acid, (ii) the shape of the c-terminal servingapparatus may be conical, the acuteness of the cone deter-mining the enzyme's sensitivity to length versus steric vol-ume, and (iii) the acyl chain is bound in a bent conformation.This latter conclusion was based on studies of C14 fatty acidshaving one double bond in either cis (Z) or trans (E) confor-mations or one triple bond (Y). Surveys of 12 tetradecynoyl-CoAs (Y2-Y13) revealed that all were substrates for Nmtlpwith the exception of 5-tetradecynoyl-CoA, which was notbound (12). 4-Tetradecynoyl-CoA and 6-tetradecynoyl-CoAwere at least as active as myristoyl-CoA. In contrast, place-ment of a cis double bond between C5 and C6 resulted in ananalog, (Z)-5-tetradecenoyl-CoA, that was a better substratethan either (Z)-4-, (Z)-6-, or (E)-5 tetradecenoyl-CoA ortetradecanoyl-CoA (12). These findings suggested that the

Abbreviation: Nmt, myristoyl-CoA:protein N-myristoyltransferase.

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bend was positioned between C5 and C6. We reasoned thatif the location of this bend is measured from the carboxylrather than methyl terminus of the acyl chain, it might bepossible to convert palmitoyl-CoA from a very poor substrateto a better one by inserting a cis double bond between C5 andC6 or by placing a rigid linear element between C6 and C7.

RESULTSSince earlier studies of tetradecynoic acids and tetradecenoicacids indicated that myristoyl-CoA had a bend between C5and C6 when bound to Nmtlp, we generated a series ofC8-C18 alkenyl and alkynyl analogs containing single doublebonds or single triple bonds, respectively, to more preciselydefine the location of the bend and to assess to what degreethe bend is measured from the carboxyl or w terminus (methylgroup) of the acyl chain. Fatty acids with a single triple bondcontain a rigid, colinear element of four carbons. A triplebond was placed between C4 and C5, C5 and C6, or C6 andC7 in C13:0, C15:0, and C16:0 to determine whether theirrelative activities as Nmtlp substrates would parallel therelative activities of Y4-, Y5-, and Y6-tetradecynoyl-CoAs.Such a result would suggest that the location of the triplebond is "measured" by the enzyme's acyl-CoA binding sitefrom the carboxyl (Cl) carbon. Inspection of the data pre-sented in Table 1 indicates that placement of the rigid, linearelement between C5 and C6 produces a profound reductionin activity (defined as the amount of analog GARASVLS-NH2 produced compared to myristoyl-GARASVLS-NH2 at30°C) in C13, C15, and C16 fatty acids just as it did withC14:0.Remarkably, movement of the triple bond one carbon

toward the carboxyl or methyl terminus (i.e., placementbetween C4 and C5 or C6 and C7) in tridecanoic, pentadec-anoic, and hexadecanoic acids not only "rescued" the loss ofactivity observed with the Y5 fatty acids but produced aconsiderable enhancement in the amount of acylpeptideproduced in the single-point assay. For C15 and C16 fattyacids, this enhancement was greatest for the Y6 compounds.For the C13 and C14 fatty acids, the Y4 and Y6 derivativeshad equivalent activities. These changes in activity could notbe simply ascribed to differences in the efficiency of conver-sion to the corresponding acyl-CoAs (Table 1 and data notshown).

Introduction of a rigid colinear element beginning at C6 inthe acyl chain of pentadecanoyl-CoA and palmitoyl-CoA(C15:Y6 and C16:Y6) increased their activities 6.5- and 5-fold(Table 1), respectively. Kinetic analysis of C16:Y6 revealedthat the change in activity reflects in large measure anincrease in peptide Vm and a resulting 10-fold increase inpeptide catalytic efficiency (Vm/Km). In contrast, there areno significant differences in the acyl-CoA Vm/Km of hexa-decanoyl-CoA and 6-hexadecynoyl-CoA (Table 2). The en-hancement of activity obtained by placing a triple bondbetween C6 and C7 becomes less pronounced over that of thecorresponding saturated acyl-CoA as chain length is reducedbelow C14 (Table 1).

Substitution of para-substituted phenylene or a 2,5-furylgroup for several methylene groups introduces a subunitwhose rigidity is comparable to that of a triple bond andwhose aggregate width is equivalent to that of four methyl-enes. We compared the activities of three compounds whoselength was equivalent to 13 carbons and contained para-substituted phenylene at C4, C5, and C6 (C13:Ar4, C13:Ar5,and C13:Ar6), two C14 fatty acids with a similar substitutionat C5 or C6 (C14:Ar5 and C14:Ar6), and four C14 equivalentanalogs with 2,5-furanyl placed at C4, C5, C6, or C7(C14:Fu4, C14:Fu5, and C14:Fu6). Their activities werecomparable to those of the corresponding tri- and tetradecy-noic acids (Table 1). Placement ofpara-phenyl or 2,5-furyl at

C5 markedly reduced activity even though the fatty acids aregood substrates for the acyl-CoA synthetase (Table 1). Theloss of activity appears to reflect their poor affinity forNmtlp, since competition experiments indicated that theseanalog CoAs produce <10%o reduction in production ofmyristoylpeptide even when present in a 100-fold molarexcess relative to myristoyl-CoA (data not shown). In con-trast, movement of the -C4H6- and <C4H20> groups to C6results in a significant enhancement of activity relative to thecorresponding tridecanoyl- and tetradecanoyl-CoAs (Table1).Table 1 documents the increase in acylpeptide production

by Nmtlp at 300C when a cis double bond is placed betweenCS and C6 of myristate and the marked reduction in activitywhen it is moved one carbon toward the carboxyl terminus[(Z)-4-tetradecenoyl-CoA has 15% ± 2% of the activity oftetradecanoyl-CoA] or two carbons toward the w terminus[(Z)-7-tetradecenoyl-CoA has 34% ± 28% of the activity].Surveys of the corresponding (E)-4-, (E)-5-, (E)-6-, and(E)-7-tetradecenoyl-CoAs revealed that the analog with atrans double bond between C5 and C6 was the least active(18% ± 1% ofC14:0-CoA), and the analog with a trans doublebond between C7 and C8 was the most active (104% ± 11%).The increase in activity observed after placing a cis double

bond between C5 and C6 of myristoyl-CoA was also ob-served in palmitoyl-CoA: with (Z)-5-hexadecenoyl-CoA(C16:Z5) as a substrate, Nmtlp generates 4 times as muchacylpeptide at 30°C in the single-point assay as with palmi-toyl-CoA and 40%o ± 7% the amount produced with myris-toyl-CoA. Kinetic analysis revealed that this change was dueprincipally to an improvement in peptide catalytic efficiency(Table 2). In contrast, no significant improvement in activitywas noted when a cis double bond was placed between C4and C5 or C6 and C7 of palmitoyl-CoA (Table 1). Moreover,(Z)-5-octenoyl-CoA, (Z)-5-decenoyl-CoA, (Z)-5-dodec-enoyl-CoA, (Z)-5-heptadecenoyl-CoA, and (Z)-S-octadec-enoyl-CoA are either less active or equivalent in their activityto the corresponding C8:0-, C10:0-, C12:0-, C17:0-, andC18:0-CoAs (Table 1).

Insertional mutagenesis of NMTI causes recessive lethal-ity, indicating that Nmtlp is essential for vegetative growth(3). One of the predictions of the ordered bi-bi reactionmechanism is that any change in the size of intracellularmyristoyl-CoA pools or in the ability ofNmtlp to gain accessto these pools should have a dramatic effect on the efficiencyof protein N-myristoylation and therefore on cell viability.nmtl -181 encodes a mutant enzyme (nmtl81p) with a Gly451-- Asp replacement, which produces a 10-fold reduction inaffinity for myristoyl-CoA at 37°C but not 24°C (15). nmtl -181strains have temperature-sensitive myristic acid auxotrophy:they will grow at the same rate as NMTJ strains in richmedium (yeast/peptone/dextrose; YPD) at 24°C but will notgrow at 37°C unless YPD medium is supplemented with -500,LM myristate. Palmitate is unable to rescue growth at 37°C,presumably reflecting the fact that the extent of its conver-sion to C14:0 by j-oxidation is insufficient to satisfy theincreased demands of nmtl-181p for myristoyl-CoA broughtabout by its Km defect.The fatty acid analogs described above represent a series

of probes that can be used to compare and contrast theacyl-CoA binding sites of Nmtlp and nmtl81p. In vitrosingle-point assays of the activities of C14 and C16 fatty acidscontaining single cis or trans double bonds, single triplebonds, or aromatic residues indicate that the overall shape ofthe acyl-CoA binding pocket is not grossly disturbed innmtl81p (Table 1). C16:ZS-CoA was 7-fold more active thanC16:0-CoA at 24°C, but equivalent at 37°C. Introduction of atriple bond between C6 and C7 in palmitate converts palmit-oyl-CoA from a poor substrate for nmtl81p (5-7% of myris-toyl-CoA at 24-370C) to one whose activity is 68% ± 23% that

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Table 1. Comparison of substrate properties of the CoA derivatives of fatty acids containing single triple bonds, cis or trans doublebonds, aromatic groups, or furanyl groups for S. cerevisiae Nmtlp and nmtl81p

Activity, % myristatetC18 HPLC Acyl-CoA

Nmt-p nmtl~lp elution time, production,Compound* 240C 300C 37C 240C 37°C % B* % C14:0-CoA

Naturally occurring fatty acidsC8:0 Octanoic acidC10:0 Decanoic acidC12:0 Dodecanoic (lauric) acidC13:0 Tridecanoic acidC14:0 Tetradecanoic (myristic) acidC15:0 Pentadecanoic acidC16:0 Hexadecanoic (palmitic) acidC17:0 Heptadecanoic acidC18:0 Octadecanoic (stearic) acid

Triple bonds (Y)C12:Y6 CH3(CH2)4CaC(CH2)4COOHC13:Y4 CH3(CH2)7GC(CH2)2COOHC13:Y5 CH3(CHA)6(>C(CH2)3COOHC13:Y6 CH3(CH2)5CSC(CH2)4COOHC14:Y4 CH3(CH2)gC=C(CH2hCOOHC14:Y5 CH3(CH2hG(aC(CH2)3COOHC14:Y6 CH3(CH2)6C(CH2)4COOHC1S:Y4 CH3(CH2)9-C(CH22COOHC1S:YS CH3(CH2)C>c(CH2)3COOHC1S:Y6 CH3(CH2),C(CH2)4COOHC16:Y4 CH3(CH2)10aC(CH2hCOOHC16:Y5 CH(CH2)~C(CH2)3COOHC16:Y6 CH3(CH2)8CC(CH2)4COOH

AromaticsC13:Ar4 CH3(CH2)6C6H4(CH2hCOOHC13:ArS CH3(CH2),C6H4(CH2)3COOHC13:Ar6 CH3(CH2)4C6H4(CH2)4COOHC13:ArlO CH3C6H4(CH2)WCOOHC13:Arll C6H5(CH2hCOOHC14:ArS CH3(CH2)5C6H4(CH2)3COOHC14:Ar6 CH3(CH2)6C6H4(CH2)4COOHC14:Arll CH3C6H4(CH2COOHC14:Arl2 C6H5(CH2)10COOH

FuransC14:Fu4 CH3(CH2)7<C4H20>(CH2)2COOHC14:FuS CH3(CH2)6<C4H2O>(CH2)3COOHC14:Fu6 CH3(CH2)5<C4H20>(CH2)4COOHC14:Fu7 CH3(CH2)4<C4H2O>(CH2)5COOH

Cis (Z) and trans (E) double bondsC8:Z5 CH3CH2CH=CH(CH2)3COOHC10:ZS CH3(CH2)3CH=CH(CH2)3COOHC12:ZS CH3(CH2)5CH=CH(CH2)3COOHC14:E4 CH3(CH2)*CH=CH(CH22COOHC14:ES CH3(CH2)CH=CH(CH2)3COOHC14:E6 CH3(CH2)6CH=CH(CH2)4COOHC14:E7 CH3(CH2)5CH=CH(CH2)5COOHC14:Z4 CH3(CH2)sCH=CH(CH2)COOHC14:Z5 CH3(CH2CH=CH(CH2)3COOHC14:Z6 CH3(CH2)6CH=CH(CH2)4COOHC14:Z7 CH3(CH2)sCH=CH(CH2)5COOHC16:Z4 CH3(CH2)1OCH=CH(CH2)2COOHC16:ZS CH3(CH2hCH=CH(CH2)3COOHC16:Z6 CH3(CH2)8CH=CH(CH2)4COOHC17:Z5 CH3(CH2)1OCH=CH(CH2)3COOHC18:ZS CH3(CH2)11CH=CH(CH2)3COOH

100§

9± 1

11 ± 4

36 ± 1249± 982 ± 8

70 ± 1810± 13 ± 0.42± 1

58 22405 5820± 4

298 ± 5489 ± 9 318 ± 15

3± 1119±20 348±65

174 ± 3215± 2

455 ± 7412± 62± 1

100 ± 20 54 ± 22

91 ± 111 ± 0.1

184 ± 1916

1147± 2

241 ± 591309

124 ± 72± 0.786± 150± 5

4± 210± 165 ± 1487 ± 3318 ± 181 ± 9104 ± 1115± 2

262 ± 9 220 ± 8999± 3734 ± 285± 0

46 ± 1 40 ± 75 ± 0.79± 0.02± 0.4

1000 1000§ lo§

4± 0.3 5± 0.1 7± 4

316 ± 15 527 ±+25<1 <1

165 ± 10 349 ± 13 1715 ± 21

55± 3 68 ± 23 23 ± 2

<1110± 1

<126± 6<1

23 217+ 1<1

105 ±21 206± 22

12± 386 ± 3959± 914± 180± 941 ± 112± 1

34± 6 67±39 12± 3

576369777882868890

66726857757272787474807776

757574747476777677

76747472

55616777767676767676748483848286

100w

39± 9

87 ± 1162 ± 8

99± 6

95 ± 10

64 ± 4

121 ± 14

101 ± 11

*Details concerning the synthesis of these compounds and their characterization by TLC, melting point, 1H NMR, 13C NMR, and MS areavailable from the authors upon request.tThe single-point discontinuous in vitro Nmtlp assay is described in ref. 12. Fatty acids were first converted to their corresponding CoAderivatives (final concentration = 18 ,uM, assuming 100%6 conversion) by using 0.3 unit ofPseudomonas acyl-CoA synthetase (Sigma) per mland a 25-min incubation at 30°C. Purified Escherichia coli-derived S. cerevisiae Nmtlp or nmtl81p (6, 14) was added (final concentration =0.04-0.08 ,g/ml for Nmtlp and 6-30 ,ug/ml for nmtl81p) together with GARE3H]ASVLS-NH2 [specific activity = 373 mCi/mmol (1 Ci = 37GBq), final concentration = 25 AM]. This well-characterized peptide substrate (12, 13) represents residues 1-8 of the N-myristoylated Pr55SaSencoded by human immunodeficiency virus 1. After a 10-min incubation at 24°C, 30°C, or 37°C, the radiolabeled acylpeptide was purified byC18 reverse-phase HPLC using gradient conditions described in ref. 12 and quantitated using an in-line scintillation counter. All assays wereperformed on at least two separate occasions, each time in triplicate. The means + SD of representative experiments are shown.tThe acylpeptide elution time from the C18 reverse-phase HPLC column is expressed as a function of the concentration of buffer B (acetonitrile+ 0.1% trifluoroacetic acid).

§For Nmtlp, 100%o at 24°C, 30°C, and 37°C = 4.8 + 0.5 x 105, 10.5 + 3.6 x 105, and 5.0 ± 0.6 x 105 pmol of myristoyl-GAR[3H]ASVLS-NH2per min per mg of enzyme, respectively. For nmtl8lp, 100% at 24°C and 37°C = 1.1 + 0.1 x 104 and 2.4 + 0.9 x 103 pmol of acylpeptide permin per mg, respectively.1The efficiency of conversion of analogs to their CoA thioesters by Pseudomonas acyl-CoA synthetase was determined by using [3H]CoA andan assay detailed in ref. 12. Assays were performed in duplicate or triplicate, and the results were compared to the amount of myristoyl-CoAproduced in a parallel reaction: [fatty acid] = 50 ,M, [CoA] = 1 mM, [acyl-CoA synthetase] = 0.03 unit/ml; 100o = 770 pmol ofmyristoyl-CoAper min.

11 + 4

..-

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Table 2. Kinetic studies of fatty acids

Corn- Peptide Acyl-CoApound Kmi, AM Vm, % Vm/Km Kmi, IM Vm, % Vm]KmC14:0 12 3 100* 9 ± 2 12 ± 5 100t 10 ± 3C14:Y6 16 4 186 ± 35 12 ± 1 8 ± 1 103 ± 5 13 ± 1C14:Z5 30 12 247 ± 55 9 ± 2 18 ± 2 218 ± 2 13 ± 1C16:0 25 8 16 ± 3 1 1 14 ± 7 12 ± 7C16:Y6 14± 6 98± 16 7±2 4±1 61±9 21± 10C16:Z5 30 8 63 ± 8 2 ± 1 3 ± 1 36 ± 1 12 ± 1The apparent peptide Km and Vm were first determined using

saturating concentrations of fatty acid. The apparent acyl-CoA Kmwas then defined using the peptide at its Km. All Vm data have beennormalized to the Vm observed in control experiments usingmyristate. Kinetic studies were performed in triplicate for eachcompound two to nine times.*100% = 4.4 x 106 ± 7 x 10W pmol of myristoyl-GARASVLS-NH2per mmn per mg of Nmtlp at 30"C (n = 6 experiments).t1O0o = 3.2 x 106 ± 1.5 x 106 pmol of acylpeptide per min per mgof enzyme at 300C (n = 4 experiments).

of myristoyl-CoA at 240C and 23% ± 2% at 370C. Remark-ably, the amount of acylpeptide produced by nmtl81p whenit was incubated with C14:Y6-CoA was 349%6 ± 13% of theamount generated with myristoyl-CoA at 240C and 1715% +21% of that at 370C. This temperature-dependent increase inacylpeptide production was not noted when nmtl81p wasincubated with the CoA derivatives of C14:0, C16:0, C16:Y6,C14:Z5, or C16:ZS; in each of these cases, the amount ofacylpeptide formed was less at 37°C than at 240C (see Table1).Given the dependency of nmtl-181 strains on exogenous

myristate for growth at the nonpermissive temperature andthe ability of its protein product to utilize C14:Z5, C14:Y6,C16:Z5, and C16:Y6 in vitro, we examined whether thesefatty acid analogs could support growth of isogenic NMT1and nmtl-181 strains at 24-370C. If these compounds weresubstrates for S. cerevisiae acyl-CoA synthetase (15) andnmtl81p in vivo, their ability to support growth could betaken as evidence that one or more of the essential N-myris-toylproteins produced by S. cerevisiae could express theirbiological functions with an unsaturated C16 rather than asaturated C14 fatty acid. An additional isogenic strain wasused for these studies that contained nmtl-72, a mutant alleleidentified during a search for conditional mutations thatrestore conjugation of haploid strains in the absence ofpheromone receptors (16). Genetic and biochemical evidencesuggests that the phenotype associated with nmtl-72 is due toreduced N-myristoylation of Gpal, a 55-kDa haploid essen-tial guanine nucleotide-binding regulatory protein a subunitinvolved in the regulatory cascade initiated when MATa andMATa cells prepare to conjugate. Metabolic labeling studiesof nmtl-181 and nmtl-72 strains using [3H]myristate reveal apattern of labeling that differs for some, but not all, cellularN-myristoylproteins at 24°C and 30WC (refs. 14 and 16; datanot shown) and suggested that Nmtlp, nmtl81p, and nmt72pmight deliver the analogs to different subsets of cellularN-myristoylproteins. Fig. 1 illustrates a typical result ob-tained when isogenic NMT1, nmtl-181, and nmtl-72 strainswere grown on YPD alone or YPD supplemented with 500,M C14:0, C14:Z5, C14:Y6, C16:0, C16:ZS, and C16:Y6 at240C, 300C, or 370C. Remarkably, C16:Y6 produced modestreductions in growth of nmtl-181, but not NMT1 and nmtl-72, strains at 24°C while C16:Z5 completely arrested growthofthe nmtl-181 strain at this temperature. In contrast, neitherC16:0, C14:Z5, nor C14:Y6 had any deleterious effect on thegrowth of cells that synthesize nmtl81p (or nmt72p andNmtlp) at this temperature. These strain-specific effects ofC16:ZS and C16:Y6 were more pronounced at 30TC. At 370C,both nmtl-181 and nmtl-72 strains showed growth inhibition

Lnrnt-/8/ nm//-72 NMT/240C 30C 370C

014-Z0C14;IZ5 m X3C14 Y6 o_

C16:0 - -

C16 Z 5 _1_03____C0 Y6

FIG. 1. Growth characteristics of isogenic S. cerevisiae strainscontaining wild-type and mutant NMT1 alleles in rich medium withor without fatty acid supplementation. Strains YM2909 (MATaNMTI ura3-52 his3A200 ade2-101 lys2-801 leu2-3,12), YB334 (nmtl-72), and YB336 (nmtl-181) were replica plated onYPD medium alone(-) or YPD supplemented with the indicated fatty acids at 500 ,uMand grown for 2 days at 24°C, 30°C, or 37C. Note that all media alsocontained 1% (wt/vol) Brij 58. The genotypes at the top of the figurecorrespond to the patches on each plate.

unless the medium was supplemented with myristate. nmtl-181 strains were partially rescued by 500 ,uM C14:Y6 at thisnonpermissive temperature.

DISCUSSIONAnalysis of the activities of C12-C16 alkynyl-CoAs andC8-C18 alkenyl-CoAs in an in vitro assay containing purifiedS. cerevisiae Nmt has provided insight about the nature oftheenzyme's acyl-CoA binding site and the structural featuresthis "host" recognizes in its bound acyl-CoA "guest."

Kinetic studies (Table 2) indicate that the enhancement ofacylpeptide production observed with C14:ZS-CoA andC14:Y6-CoA compared to C14:0-CoA reflect an improve-ment in peptide Vm/Km that is greater than the improvementin acyl-CoA Vm/Km. Given Nmtlp's ordered bi-bi reactionmechanism and the cooperative interactions known to occurbetween its acyl-CoA and peptide binding sites, we interpretthis finding to mean that these conformationally restrictedanalog CoAs optimize Nmtlp's peptide binding site forinteraction with its substrate and/or promote catalytic trans-fer of acyl chain to peptide by more favorable positioning ofthe acyl-CoA thioester bond for subsequent hydrolysis. Mo-lecular models together with the results presented in Tables1 and 2 suggest that the increases in acylpeptide productionobserved with C12-C15 alkynyl-CoAs containing a triplebond between C4 and C5 or C6 and C7 reflect a binding sitegeometry that is more restrictive in the region that interactswith Cl-CS ofmyristoyl-CoA than in the region that interactswith C6-C14.The enhancement in acylpeptide production observed

when a cis double bond is placed between C5 and C6 in theacyl chains of C12:0- to C17:0-CoAs but not C8:0-, C10:0-,and C18:0-CoAs appears likely to reflect a combination offactors: (i) the distance between C1 and a bend located nearC5 is a critical determinant for optimal positioning of theacyl-CoA guest in the binding site of the Nmtlp host, and (ii)the length of the acyl chain between the bend and the cterminus has an important effect on the stability of the

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host-guest interaction and/or positioning of the guest in theacyl-CoA binding site. The acyl-CoA Vm/Km of palmitoyl-CoA is comparable to myristoyl-CoA, whereas its peptidecatalytic efficiency is considerably worse. The enhancementof activity observed with C16:Z5-CoA (and C16:Y6-CoA)appears to be due to an improvement in peptide Vm/Km (seeTable 2). This phenomenon may reflect (i) palmitoyl-CoA'sinability to assume a conformation in the acyl-CoA bindingsite that mimics that ofmyristoyl-CoA, (ii) the ability ofthesestrategically placed double and triple bonds to create aconformationally restricted 16-carbon acyl chain structurethat "reproduces" that of myristoyl-CoA at least from Cl-C5, and/or (iii) a "spring-loading" effect, produced byinterposing two additional methylenes between the "bend"and the w-terminal sensor, that subsequently facilitates as-sumption of the transition state. These considerations mayhave impact on the design of transition-state inhibitors orinhibitors that can mimic the spatial relationships of boundacyl chain, CoA, and peptide. Heteronuclear NMR methodsshould allow direct determination of the conformation ofstable, isotope-enriched, fatty acid analog CoAs bound toNmtlp.The ability of C14:Y6 to partially rescue growth of two

isogenic strains of S. cerevisiae that contain distinct nmtlmutations resulting in temperature-sensitive myristic acidauxotrophy is consistent with its enhanced activity relative tomyristoyl-CoA in the in vitro assay system. Such rescuesuggests that 6-tetradecynoic acid (C14:Y6) (i) is efficientlyimported in this yeast, (ii) is activated by S. cerevisiaeacyl-CoA synthetase Faalp or Faa2p (16), (iii) is incorpo-rated into critical N-myristoylproteins, and (iv) does notprevent expression ofthe biological function ofthese proteinsor that its triple bond is saturated prior to, or after, use as anNmtlp substrate. The failure of C14:Z5 to rescue growth ofstrains containing nmtl-72 or nmtl-181 at the nonpermissivetemperature (together with its lack of effect on the growth ofan isogenic NMTI strain) indicates that this excellent sub-strate is not able to satisfy one or more of criteria i-iv. C16:Z5and C16:Y6 have no effect on the growth ofNMT1 strains at24-37°C and do not rescue the growth arrest produced bynmtl-72 and nmtl-181 on YPD media at 37°C, suggesting thatneither analog is imported, activated to its CoA thioester,and/or metabolically processed to the corresponding C14:Z5or C14:Y6 derivatives at efficiencies necessary to overcomethe defects in nmtl81p and nmt72p revealed at this temper-ature. Surprisingly, these two analogs completely (C16:Z5) orpartially (C16:Y6) suppress growth of an nmtl-181 strain atthe permissive temperature (24°C). It is important to note thatthis effect is analog- and strain-specific and that C16:0 atidentical concentrations does not affect the growth of any ofthe three isogenic strains at 24°C. C16:Z5 and C16:Y6 havesimilar substrate properties in vitro for nmtl81p and Nmtlpat 24°C and are comparable to myristoyl-CoA (Table 1).Thus, the ability of these compounds to produce growtharrest in nmtl-181 strains could reflect (i) their greaterrepresentation in critical N-myristoylproteins in strains pro-

ducing nmtl81p compared to nmt72p or Nmtlp, together withthe fact that these proteins require C14 fatty acids forexpression of biological activity, and/or (ii) their ability toproduce absolute or relative reductions in myristoyl-CoApools used by nmtl81p so that the level of protein N-myris-toylation is reduced below some critical level compatible withvegetative growth. Direct proof of i will require synthesis ofradiolabeled C16:Z5 or C16:Y6 for metabolic labeling stud-ies. Nonetheless, identification of conformationally re-stricted analogs of palmitate that are substrates for wild-typeor mutant Nmts offers an opportunity to convert this myris-toyltransferase to a palmitoyltransferase. By delivering (16fatty acids to N-myristoylproteins in vivo, we should be ableto begin to assess why C14:0 rather than unsaturated orlonger acyl chains were "selected" for this cotranslational,covalent protein modification.

We thank Steve Reed for supplying us with the nmtl-72 mutantallele. This work was supported by Grants A127179 and A130188from the National Institutes of Health and from Monsanto.

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