Modulation of recombinant human prostate-specific antigen: activation by Hofmeister salts and inhibition by azapeptides. Appendix: thermodynamic interpretation of the activation by concentrated salts.

Prostate specific antigen (PSA, also known as human kallikrein 3) is an important diagnostic indicator of prostatic disease. PSA exhibits low protease activity (>10(4)-fold less than chymotrypsin) under the usual in vitro assay conditions. In addition, PSA does not react readily with prototypical serine protease inactivators. We expressed human PSA (rh-PSA) in Escherichia coli and have demonstrated that rh-PSA has properties similar to those of native PSA isolated from human seminal fluid. Both PSA and rh-PSA are >10(3)-fold more active in the presence of 1.3 M Na(2)SO(4). This activation is anion-dependent, following the Hofmeister series when normality is considered: SO(4)(2)(-) approximately citrate > Ac(-) > Cl(-) > Br(-) > I(-). The nature of the cation has little effect on salt activation. The rate of inactivation of rh-PSA by DFP is 30-fold faster in the presence of 0.9 M Na(2)SO(4), and the rate of inactivation by Suc-Ala-Ala-Pro-Phe-CK is >20-fold faster under these conditions. Azapeptides containing Phe or Tyr at position P(1) also inactivate rh-PSA in the presence of high salt concentrations. These compounds represent the first described inhibitors designed to utilize the substrate binding subsites of PSA. CD spectroscopy demonstrates that the conformation of rh-PSA changes in the presence of high salt concentrations. Analytical ultracentifugation and dynamic light scattering indicate that PSA remains monomeric under high-salt conditions. Interestingly, human prostatic fluid contains as much as 150 micro mol citrate/g wet weight, which suggests that salt concentrations may regulate PSA activity in vivo.

Mechanistic studies of two dioxygenases in the methionine salvage pathway of Klebsiella pneumoniae.

Two dioxygenases (ARD and ARD') were cloned from Klebsiella pneumoniae that catalyze different oxidative decomposition reactions of an advanced aci-reductone intermediate, CH(3)SCH(2)CH(2)COCH(OH)=CH(OH) (I), in the methionine salvage pathway. The two enzymes are remarkable in that they have the same polypeptide sequence but bind different metal ions (Ni(2+) and Fe(2+), respectively). ARD converts I to CH(3)SCH(2)CH(2)COOH, CO, and HCOOH. ARD' converts I to CH(3)SCH(2)CH(2)COCOOH and HCOOH. Kinetic analyses suggest that both ARD and ARD' have ordered sequential mechanisms. A model substrate (II), a dethio analogue of I, binds to the enzyme first as evidenced by its lambda(max) red shift upon binding. The dianion formation from II causes the same lambda(max) red shift, suggesting that II bind to the enzyme as a dianion. The electron-rich II dianion likely reacts with O(2) to form a peroxide anion intermediate. Previous (18)O(2) and (14)C tracer experiments established that ARD incorporates (18)O(2) into C(1) and C(3) of II and C(2) is released as CO. ARD' incorporates (18)O(2) into C(1) and C(2) of II. The product distribution seems to necessitate the formation of a five-membered cyclic peroxide intermediate for ARD and a four-membered cyclic peroxide intermediate for ARD'. A model chemical reaction demonstrates the chemical and kinetic competency of the proposed five-membered cyclic peroxide intermediate. The breakdown of the four-membered and five-membered cyclic peroxide intermediates gives the ARD' and ARD products, respectively. The nature of the metal ion appears to dictate the attack site of the peroxide anion and, consequently, the different cyclic peroxide intermediates and the different oxidative cleavages of II. A cyclopropyl substrate analogue inactivates both enzymes after multiple turnovers, providing evidence that a radical mechanism may be involved in the formation of the peroxide anion intermediate.

Inactivation of cysteine proteases by (acyloxy)methyl ketones using S'-P' interactions.

We have synthesized (acyloxy)methyl ketone inactivators of papain, cathepsin B, and interleukin-1beta conversion enzyme (ICE) that interact with both the S and S' subsites. The value of k(inact)/K(i) for these inactivators is strongly dependent on the leaving group. For example, Z-Phe-Gly-CH(2)-X is a poor inactivator of papain when X is OCOCH(3) (k(inact)/K(i) = 2.5 M(-)(1) s(-)(1)) but becomes a potent inactivator when X is OCO-L-Leu-Z (k(inact)/K(i) = 11 000 M(-)(1) s(-)(1)). Since these leaving groups have similar chemical reactivities, the difference in potency must be attributed to interactions with the S' sites. The potency of the leaving group correlates with the P' specificity of papain. Similar results are also observed for the inactivation of cathepsin B by these compounds. A series of inactivators with the general structure Fmoc-L-Asp-CH(2)-X were designed to inactivate ICE. No inhibition was observed when X was OCOCH(3). In contrast, ICE is inactivated when X is OCO-D-Pro-Z (k(inact)/K(i) = 131 M(-)(1) s(-)(1)). These results demonstrate that S'-P' interactions can be utilized to increase the efficacy and selectivity of (acyloxy)methyl ketone inactivators.

One protein, two enzymes.

Two enzymes, designated, E-2 and E-2', catalyze different oxidation reactions of an aci-reductone intermediate in the methionine salvage pathway. E-2 and E-2', overproduced in Escherichia coli from the same gene, have the same protein component. E-2 and E-2' are separable on an anion exchange column or a hydrophobic column. Their distinct catalytic and chromatographic properties result from binding different metals. The apo-enzyme, obtained after metal is removed from either enzyme, is catalytically inactive. Addition of Ni2+ or Co2+ to the apo-protein yields E-2 activity. E-2' activity is obtained when Fe2+ is added. Production in intact E. coli of E-2 and E-2' depends on the availability of the corresponding metals. These observations suggest that the metal component dictates reaction specificity.

Inactivation of cysteine proteases.

The cysteine proteases papain and cathepsin B are inactivated by a Michael acceptor, a peptidyl-beta-chloro-alpha, beta-unsaturated ester (N-Ac-L-Phe-NHCH2-CCl=CH-COOMe). Inactivation occurred concomitant with chloride release which was stoichiometric with the amount of enzyme. This result is consistent with nucleophilic attack of the active site cysteine on the beta-carbon of the inhibitor, followed by expulsion of chloride ion. Inactivation by this class of compounds requires the carbon skeleton about the double bond to be in the trans configuration. The cis isomer was a competitive inhibitor. The difference in the mode of inhibition between the isomers is probably due to non-productive binding of the cis isomer due to bulky chlorine substituent in the beta-position.

Inhibitors of human heart chymase based on a peptide library.

We have synthesized two sets of noncleavable peptide-inhibitor libraries to map the S and S' subsites of human heart chymase. Human heart chymase is a chymotrypsin-like enzyme that converts angiotensin I to angiotensin II. The first library consists of peptides with 3-fluorobenzylpyruvamides in the P1 position. (Amino acid residues of substrates numbered P1, P2, etc., are toward the N-terminal direction, and P'1, P'2, etc., are toward the C-terminal direction from the scissile bond.) The P'1 and P'2 positions were varied to contain each one of the 20 naturally occurring amino acids and P'3 was kept constant as an arginine. The second library consists of peptides with phenylalanine keto-amides at P1, glycine in P'1, and benzyloxycarbonyl (Z)-isoleucine in P4. The P2 and P3 positions were varied to contain each of the naturally occurring amino acids, except for cysteine and methionine. The peptides of both libraries are attached to a solid support (pins). The peptides are evaluated by immersing the pins in a solution of the target enzyme and evaluating the amount of enzyme absorbed. The pins with the best inhibitors will absorb most enzyme. The libraries select the best and worst inhibitors within each group of peptides and provide an approximate ranking of the remaining peptides according to Ki. Through this library, we determined that Z-Ile-Glu-Pro-Phe-CO2Me and (F)-Phe-CO-Glu-Asp-ArgOMe should be the best inhibitors of chymase in this collection of peptide inhibitors. We synthesized the peptides and found Ki values were 1 nM and 1 microM, respectively. The corresponding Ki values for chymotrypsin were 10 nM and 100 microM. The use of libraries of inhibitors has advantages over the classical method of synthesis of potential inhibitors in solution: the libraries are reusable, the same libraries can be used with a variety of different serine proteases, and the method allows the screening of hundreds of compounds in short periods of time.

The methionine salvage pathway in Klebsiella pneumoniae and rat liver. Identification and characterization of two novel dioxygenases.

The 5-methylthio-D-ribose moiety of 5'-(methylthio)-adenosine is converted to methionine in a wide variety of organisms. 1,2-Dihydroxy-3-keto-5-methylthiopentene anion (an aci-reductone) is an advanced intermediate in the methionine salvage pathway present in the Gram-negative bacterium Klebsiella pneumoniae and rat liver. This metabolite is oxidized spontaneously in air to formate and 2-keto-4-methylthiobutyric acid (the alpha-keto acid precursor of methionine). Previously, we had purified an enzyme (E2) from Klebsiella which catalyzes the oxidative degradation of the aci-reductone to formate, CO, and methylthiopropionic acid. To further characterize the reactions of the aci-reductone we used its desthio analog, 1-2-dihydroxy-3-ketohexene anion (III), which was described previously. This molecule undergoes the analogous enzymatic and non-enzymatic reactions of the natural substrate, namely the formation of formate, CO, and butyrate from III. Experiments with 18O2 show that E2 is a dioxygenase which incorporates one molecule of 18O into formate and butyric acid. No cofactor has been identified. We were unable to find an enzyme which catalyzes the conversion of 1,2-dihydroxy-3-keto-5-methylthiopentane to a keto acid precursor of methionine. The keto acid is probably produced non-enzymically in Klebsiella. We have, however, identified and purified an enzyme (E3) from rat liver, which catalyzes the formation of formate and 2-oxopentanoic acid from III. This enzyme has a monomeric molecular mass of 28,000 daltons, and no chromophoric cofactor has been identified. Experiments with 18O2 show that E3 is a dioxygenase which incorporates an 18O molecule into formate and the alpha-keto acid. In rat liver CO formation was not detected.

Mechanism of mevalonate pyrophosphate decarboxylase: evidence for a carbocationic transition state.

Mevalonate pyrophosphate decarboxylase catalyzes the decarboxylation of mevalonate pyrophosphate to isopentyl pyrophosphate. The mechanism of action of this enzyme was investigated to elucidate the mechanism of inhibition by 3-hydroxy-3-(fluoromethyl)-5-pyrophosphopentanoic acid (II). It was previously found that II is a competitive inhibitor (Ki = 0.01 microM) of the enzyme reaction [Reardon, J.E., & Abeles, R.H. (1987) Biochemistry 26, 4717-4722; Nave, J.F., d'Orchymont, H., Ducep, J.B., Piriou, F., & Jung, M.J. (1985) Biochem. J. 227, 247-254]. We have now observed that II is decarboxylated 2500-fold more slowly than mevalonate pyrophosphate (3-hydroxy-3-methyl-5-pyrophosphopentanoic acid, I). The enzyme was exposed to saturating concentrations of II and ATP and then passed through a Penefsky column to remove excess substrate. The enzyme was denatured immediately upon emerging from the Penefsky column. Nearly 1 equiv of both 3-phospho-3-(fluoromethyl)-5-pyrophosphopentanoic acid and ADP was bound to the enzyme. 3-Hydroxy-5-pyrophosphopentanoic acid (III) is phosphorylated at the secondary hydroxyl group and released from the enzyme without decarboxylation. This reaction is 30-fold slower than the rate of decarboxylation of I. The introduction of the 3-fluoromethyl group as well as the removal of the 3-methyl group results in low rates of decarboxylation. These substrate analogs have decreased electron density relative to the tertiary carbon of the substrate. Therefore, the transition state of the decarboxylation step has considerable carbocationic character. Further support for the carbocationic transition state is provided by the finding that N-methyl-N-carboxymethyl-2-pyrophosphoethanolamine (IV) inhibits the enzyme reaction with Ki = 0.75 microM. IV is probably a transition-state analog in which the positively charged nitrogen atom is analogous to the carbocation.

Three-dimensional structure of chymotrypsin inactivated with (2S)-N-acetyl-L-alanyl-L-phenylalanyl alpha-chloroethane: implications for the mechanism of inactivation of serine proteases by chloroketones.

The reaction of enantiomerically pure (2S)-N-acetyl-L-alanyl-L-phenylalanyl alpha-chloroethane with gamma-chymotrypsin was studied as a probe of the mechanism of inactivation of serine proteases by peptidyl chloroalkanes. It was determined crystallographically that the peptidyl chloroethane alkylates His57 with retention of configuration at the chiral center, indicating a double displacement mechanism. We think it likely that a Ser195-epoxy ether adduct is an intermediate on the inactivation pathway, although other possibilities have not been disproven. Kinetic data reported by others [Angliker et al. (1988) Biochem. J. 256, 481-486] indicate that the epoxy ether intermediate is not an irreversibly inactivated form of enzyme [a conclusion confirmed experimentally (Prorok et al. (1994) Biochemistry 33, 9784-9790)] and that both ring closure of the tetrahedral intermediate to form the epoxy ether and ring opening by His57 partially limit the first-order rate constant for inactivation, ki. The peptidyl chloroethyl derivative adopts a very different active site conformation from that assumed by serine proteases inactivated by peptidyl chloromethanes. Positioning the chloroethyl derivative into the conformation adopted by chloromethyl derivatives would cause the extra methyl group to make a bad van der Waals contact with the inactivator P2 carbonyl carbon, thereby preventing the formation of the invariant hydrogen bond between the inactivator P1 amide nitrogen and the carbonyl group of Ser214. We conclude that the unusual conformation displayed by the chloroethyl derivative is caused by steric hindrance between the extra methyl group and the rest of the inactivator chain.

Chloroketone hydrolysis by chymotrypsin and N-methylhistidyl-57-chymotrypsin: implications for the mechanism of chymotrypsin inactivation by chloroketones.

We have examined the reaction of N-(benzyloxycarbonyl)-L-alanyl-L-glycyl-L-phenylalanyl chloromethyl ketone (ZAGFCMK) with chymotrypsin (Cht) and have found that, in addition to irreversible alkylation of the enzyme, some of the corresponding hydroxymethyl ketone is produced. For each molecule of hydroxy ketone formed, 3.6 molecules of chymotrypsin are inactivated. Chloroketone hydrolysis is also observed with chymotrypsin methylated at N-3 of the active site histidine (MeCht). The hydrolysis proceeds slowly (k = 0.14 min-1). Alkylation of the modified enzyme was not observed. An initial burst of free chloride is detected during the MeCht-catalyzed hydrolysis. The magnitude of the chloride burst is proportional to the enzyme concentration in an approximate 1:1 stoichiometry and indicates a relatively rapid chloride-releasing step which gives rise to an intermediate which is more slowly converted to hydroxy ketone. We have also investigated both the solution and MeCht-mediated hydrolysis of the S isomer of N-acetyl-L-alanyl-L-phenylalanyl chloroethyl ketone (S-AcAFCEK). We have concluded that the nonenzymatic hydrolysis proceeds with inversion of configuration at the stereocenter, while the enzymatic process occurs with retention of configuration. The two nucleophilic displacements attending the MeCht-mediated hydrolysis of S-AcAFCEK imply the formation of an intermediate, possibly of an epoxy ether, formed by internal displacement of the chloride by the oxyanion of the initially generated enzyme-chloroketone hemiketal adduct.

The fate of the carboxyl oxygens during D-proline reduction by clostridial proline reductase.

D-Proline is converted to 5-amino valeric acid by D-proline reductase. This conversion involves the reductive cleavage of the alpha-carbon-nitrogen bond. We have examined the fate of the carboxyl oxygen atoms during conversion of D-proline to delta-NH2-valeric acid. 18O atoms from the carboxyl group of D-proline are not lost during conversion to product. In contrast, in the conversion of glycine to acetyl phosphate by glycine reductase a carboxyl oxygen atom is lost to solvent. An intermediate acyl-enzyme is found during the reduction of glycine. We conclude that the reduction of proline proceeds without the formation of an acyl enzyme intermediate.

Purification and characterization of an enzyme involved in oxidative carbon-carbon bond cleavage reactions in the methionine salvage pathway of Klebsiella pneumoniae.

The 5-methylthio-D-ribose moiety of 5'-(methylthio)-adenosine is converted to methionine in a wide variety of organisms. 2,3-Diketo-5-methylthio-1-phosphopentane is an advanced intermediate in the methionine recycling pathway present in the Gram-negative bacterium Klebsiella pneumoniae. This unusual metabolite is oxidatively cleaved to yield formate (from C-1), 2-keto-4-methylthiobutyrate (the transamination product of methionine), and 3-methylthiopropionate. To further characterize this oxidative conversion, the desthio analog of the naturally occurring diketone, namely 2,3-diketo-1-phosphohexane I, was synthesized. If the metabolism of I is analogous to that of 2,3-diketo-5-methylthio-1-phosphopentane it should be converted to formate, 2-ketopentanoate, and butyrate. An enzyme (E-1), which mediates the oxidative conversion of I to formate and 2-ketopentanoate, was isolated from extracts of K. pneumoniae. E-1 was purified 100-fold to homogeneity in 10% yield. The native enzyme is a monomeric protein of M(r) 27,000. The activity of E-1 requires magnesium ion as a cofactor. No other prosthetic groups were detected. Incubation of the enzyme with I, under anaerobic conditions, led to the discovery of two intermediates. These species have been identified by 1H and 13C NMR, UV-visible spectroscopy, and model chemistry studies as 2-hydroxy-3-keto-1-phospho-1-hexene II, generated by enolization of I; and 1,2-dihydroxy-3-keto-1-hexene III, generated by enzymatic dephosphorylation of II. Intermediates II and III are released from the active site of the enzyme; III accumulates under anaerobic conditions. Under aerobic conditions, III is non-enzymically oxidized to 2-ketopentanoate, formate, and other products. Compound II was also generated by heating I at pH 7.5 for 7 min. Action of alkaline phosphatase on II produces III.

A bacterial enzyme that catalyzes formation of carbon monoxide.

We have isolated and purified an enzyme (E-2) from Klebsiella pneumoniae, which catalyzes the formation of CO from CH3-S-CH2-CH2-CO-C(OH) = CH-O- (III). This compound is an intermediate in the conversion of 5'-methylthioadenosine to methionine. Concomitant with CO formation, methylthiopropionic acid and formate are produced and O2 is consumed. E-2 also catalyzes the formation of CO, formate, and butyrate from CH3-CH2-CH2-CO-C(OH) = CH-O- (IIIa), the desthio analog of III. Experiments with isotopic IIIa have shown that formate is derived from 1-C, and CO from 2-C. E-2 has a M(r) = 18,500 and requires Mg2+, and no chromophoric cofactor has been detected.

Inhibition of tryptophan synthase by (1-fluorovinyl)glycine.

Tryptophan synthase (alpha 2 beta 2 complex) from Salmonella typhimurium catalyzes the formation of tryptophan from serine and indole. The enzyme is inactivated by (1-fluorovinyl)glycine. Concomitant with enzyme inactivation, the absorbance at 485 nm increases, indicating covalent modification of pyridoxal 5'-phosphate. It is proposed that inactivation involves elimination of HF to form an allene, which reacts with a nucleophile at the active site. The inactivation reaction involves an alpha,beta-elimination, as does the formation of tryptophan from indole and serine. The inactivation occurs with k(in) > 1.3 s-1, which is very close to k(cat) (6.4 s-1) for the formation of tryptophan from indole and serine. The inactive enzyme (alpha 2 beta 2) regains activity with k(off) = 0.005 min-1. Aminoacetone is formed during reaction, and pyridoxal 5'-phosphate is regenerated. Tryptophan synthase also catalyzes the dehydration of serine, or 3-fluoroalanine, to pyruvate in the absence of indole. This reaction involves an alpha,beta-elimination and the intermediate formation of an aminoacrylate adduct with pyridoxal 5'-phosphate, as does the formation of tryptophan. Pyruvate formation proceeds at less than 5% the rate of tryptophan formation. With [2-2H]serine an isotope effect (DVmax = 1.5) is observed. We propose that pyruvate formation is limited by the rate of hydration of the aminoacrylate intermediate and the rate of the abstraction of the serine alpha-hydrogen.

Cysteine protease inhibition by azapeptide esters.

Papain, a prototype cysteine protease, was inhibited in a time-dependent manner by azapeptide esters designed to deliver an azaglycine group to the active-site thiol. For example, the rate of inhibition was 18 M-1s-1 for Ac-L-PheAglyOiBu (2) and > 11,000 M-1s-1 for Ac-L-PheAglyOPh (7). The rate of inhibition was slowed in the presence of substrate, and there was no reactivation of the inhibited enzyme after dialysis and incubation in the assay buffer. The inhibited enzyme was completely reactivated after the addition of valine methyl ester. The inhibited form of the enzyme is presumed to be acylated on the active-site thiol. An azaalanine-based peptide inhibited papain much more slowly. Azapeptide alkyl esters are unreactive with serine proteases; therefore, these inhibitors are selective for cysteine proteases.

Role of serine 214 and tyrosine 171, components of the S2 subsite of alpha-lytic protease, in catalysis.

The function of a hydrogen bond network, comprised of the hydroxyl groups of Tyr 171 and Ser 214, in the hydrophobic S2 subsite of alpha-lytic protease, was investigated by mutagenesis and the kinetics of a substrate analog series. To study the catalytic role of the Tyr 171 and Ser 214 hydroxyl groups, Tyr 171 was converted to phenylalanine (Y171F) and Ser 214 to alanine (S214A). The double mutant (Y171F: S214A) also was generated. The single S214A and double Y171F:S214A mutations cause differential effects on catalysis and proenzyme processing. For S214A, kcat/Km is (4.9 x 10(3))-fold lower than that of wild type and proenzyme processing is blocked. For the double mutant (Y171F:S214A), kcat/Km is 82-fold lower than that of wild type and proenzyme processing occurs. In Y171F, kcat/Km is 34-fold lower than that of wild type, and the proenzyme is processed. The data indicate that Ser 214, although conserved among serine proteases and hydrogen bonded to the catalytic triad [Brayer, G. D., Delbaere, L. T. J., & James, M. N. G. (1979) J. Mol. Biol. 131, 743], is not essential for catalytic function in alpha-lytic protease. A substrate series (in which peptide length is varied) established that the mutations (Y171F and Y171F:S214A) do not alter enzyme-substrate interactions in subsites other than S2. The pH dependence of kcat/Km for Y171F and Y171F:S214A has changed less than 0.5 unit from that of wild type; this suggests the catalytic triad is unperturbed. In wild type, hydrophobic interactions at S2 increase kcat/Km by up to (1.2 x 10(3))-fold with no effect on Km.(ABSTRACT TRUNCATED AT 250 WORDS)

Inhibition of chymotrypsin by fluorinated alpha-keto acid derivatives.

A series of fluorinated alpha-keto acid derivatives [PhCHFCOCO2R,PhCH2CHFCOCO2R,PhCF2-COCO2R, and PhCH2CF2COCO2R (R = H, Me, and Et)] was synthesized. They were inhibitors of chymotrypsin, with Ki values ranging from 4700 to 15 microM. Benzylpyruvic derivatives were generally more potent than the corresponding phenylpyruvic analogs. Esters of the first series were also more potent than their corresponding acids, and potency increased with the number of fluorine atoms. By replacing the ethoxy group of PhCH2CF2COCO2Et (15b) with an amino acid chain (i.e., alanyl-leucyl-arginine methyl ester hydrochloride and alanyl-leucyl-valine ethyl ester), the resultant peptides PhCH2CF2COCO-Ala-Leu-Arg-OMe.HCl.H2O (20) and PhCH2CF2COCO-Ala-Leu-Val-OEt.H2O (23) were found to be slow-binding inhibitors of chymotrypsin with considerably lower Ki values (0.19 and 3.6 microM, respectively). 19F NMR studies indicate, in the case of 20, the presence of an enzyme-inhibitor complex with a hemiketal structure similar to those observed between trifluoromethyl ketones and chymotrypsin. The results illustrate that effective protease inhibitors can be designed by enhancing the electrophilic character of the reactive carbonyl group (with an electron-withdrawing group placed on each side of the carbonyl group). Their potency and/or selectivity can also be improved by taking advantage of binding interactions at S' subsites of the protease.

Mechanism of action of clostridial glycine reductase: isolation and characterization of a covalent acetyl enzyme intermediate.

Clostridial glycine reductase consists of proteins A, B, and C and catalyzes the reaction glycine + Pi + 2e(-)----acetyl phosphate + NH4+. Evidence was previously obtained that is consistent with the involvement of an acyl enzyme intermediate in this reaction. We now demonstrate that protein C catalyzes exchange of [32P]Pi into acetyl phosphate, providing additional support for an acetyl enzyme intermediate on protein C. Furthermore, we have isolated acetyl protein C and shown that it is qualitatively catalytically competent. Acetyl protein C can be obtained through the forward reaction from protein C and Se-(carboxymethyl)selenocysteine-protein A, which is generated by the reaction of glycine with proteins A and B [Arkowitz, R. A., & Abeles, R. H. (1990) J. Am. Chem. Soc. 112, 870-872]. Acetyl protein C can also be generated through the reverse reaction by the addition of acetyl phosphate to protein C. Both procedures lead to the same acetyl enzyme. The acetyl enzyme reacts with Pi to give acetyl phosphate. When [14C]acetyl protein C is denaturated with TCA and redissolved with urea, radioactivity remained associated with the protein. At pH 11.5 radioactivity was released with t1/2 = 57 min, comparable to the hydrolysis rate of thioesters. Exposure of 4 N neutralized NH2OH resulted in the complete release of radioactivity. Treatment with KBH4 removes all the radioactivity associated with protein C, resulting in the formation of [14C]ethanol. We conclude that a thiol group on protein C is acetylated. Proteins A and C together catalyze the exchange of tritium atoms from [3H]H2O into acetyl phosphate.(ABSTRACT TRUNCATED AT 250 WORDS)