The slow, tight binding of bestatin and amastatin to aminopeptidases

Bestatin reversibly inhibits Aeromonas aminopeptidase (EC 3.4.11.10) in a process that is remarkable for its unusual degree of time dependence. The binding of bestatin by both Aeromonas aminopeptidase and cytosolic leucine aminopeptidase (EC 3.4.11.1) is slow and tight, with Ki values (determined from rate constants) of 1.8 X 10(-8) and 5.8 X 10(-10) M, respectively. In contrast, microsomal aminopeptidase (EC 3.4.11.2) binds bestatin in a rapidly reversible process with a Ki value of 1.4 X 10(-6) M. Kinetic analysis of the slow inhibition observed is facilitated by the use of a variety of experimental treatments, primarily measurements made during pre-equilibrium; however, careful selection of conditions permits use also of steady state observations. When titrated with bestatin, 1 mol of cytosolic leucine aminopeptidase (containing 6 g atoms each of zinc and manganese) is rendered 80% inactive by 1 mol of inhibitor, thus suggesting that enzymatic activity depends on one active site/hexamer; titration of Aeromonas aminopeptidase by bestatin reveals a 1:1 stoichiometry. Amastatin inhibits all three aminopeptidases through the mechanism of slow, tight binding with Ki values ranging from 3.0 X 10(-8) to 2.5 X 10(-10) M. This behavior of microsomal aminopeptidase contrasts sharply with its rapidly reversible inhibition by bestatin. The slow, tight binding observed with five of the six aminopeptidase-inhibitor pairs investigated suggests the formation of a transition state analog complex between the enzyme and inhibitor. Physical evidence consistent with this possibility was provided by the observation that both bestatin and amastatin perturb the absorption spectrum of cobalt Aeromonas aminopeptidase.     

Inhibition of aminopeptidases by aminophosphonates

More than 30 aminophosphonates were synthesized to probe how the structural changes introduced into the phosphonic acid analogue of leucine, a potent inhibitor of cytosolic leucine aminopeptidase (Giannousis & Bartlett, 1987), affect their ability to inhibit cytosolic (EC 3.4.11.1) and microsomal (EC 3.4.11.2) aminopeptidases. Although most of the compounds studied were found to exert only a modest competitive inhibitory effect, nearly every modification of the structure of the phosphonic acid analogue of leucine was reflected in a marked difference in the affinities of these compounds for the two enzymes. [1-Amino-2-(N-alkylamino)ethyl]phosphonic acids are effective inhibitors of the microsomal enzyme, acting in a time-dependent manner. Kinetic data obtained for these inhibitors correspond to the mechanism for a biphasic slow-binding inhibition process: E + I in equilibrium E* in equilibrium E*I, in which the slow initial isomerization of the enzyme is followed by the fast formation of enzyme-inhibitor complex. The most effective inhibitor of this type was [1-amino-2-(N-cyclohexylamino)ethyl]phosphonic acid, which has a Ki value of 0.87 microM toward the microsomal aminopeptidase--a value that can be considered as equipotent with bestatin and with leucinal and hydroxamic acids, the strongest known nonpeptide inhibitors of this enzyme.     

alpha-Aminoboronic acid derivatives: effective inhibitors of aminopeptidases

alpha-Aminoboronic acids and their derivatives have been synthesized as stable white solids. These compounds are effective inhibitors of human enkephalin degrading aminopeptidase, microsomal leucine aminopeptidase (EC 3.4.11.2), and cytosolic leucine aminopeptidase (EC 3.4.11.1) at micro- to nanomolar concentrations. The inhibition of cytosolic leucine aminopeptidase has been studied in some detail. Kinetic data correspond to the mechanism for biphasic slow-binding inhibition: E + I in equilibrium E.I in equilibrium E.I*, in which a rapid initial binding is followed by a slow transformation to a stable enzyme inhibitor complex. The initial and final binding constants are dependent on the nature of the side chain at the alpha-carbon atom but are independent of the protecting group on the boronic acid moiety and follow the trend for the hydrolysis of the corresponding amino acid amides. The first-order rate constant for the transformation of E.I to E.I* is similar for all four compounds studied. These data suggest that the slow-binding step represents the formation of tetrahedral boronate species from trigonal boronic acid.     

Purification and characterization of leucine aminopeptidase from kidney bean cotyledons

A leucine aminopeptidase (EC 3,4,11.1) was purified from cotyledons of resting kidney beans ( Phaseolus vulgaris L. cv. Processor) by acidic extraction, ammonium sulfate fractionation and chromatography on DEAE-Sephacel, Sephacryl S-300, Mono Q HPLC and Superose HPLC columns. The yield of the 317-fold purified enzyme was 9%. On gel filtrations on Sephacryl S-300 and Superose HPLC the elution volumes of the enzyme corresponded to an M, of 360 000. The enzyme gave one band on native gel electrophoresis and an electrophoretic titration in an immobilized pH gradient gave a single curve with a pI of 4.8. Two bands were observed in an SDS-gel electrophoresis with M_r values of 58 000 and 60 000 both with and without reduction by 2-mercaptoethanol, indicating that subunits of the enzyme are not linked by disulphide bridges. The purified enzyme most rapidly liberated Leu and Ala of the N-termini of di-and oligopeptides, optimally at pH 9.0 ± 0.5. The enzyme was stable in the presence of glycerol, dithiothreitol and Mg²⁺, while the latter also had an activating effect. Bestatin inhibited the enzyme competitively with Leu-Gly-Gly with a Kⁱ-value of 1.5 nM . These observations indicate that the purified aminopeptidase from the cotyledons of resting kidney beans corresponds to the cytosolic leucine aminopeptidase of mammalian tissues (EC 3.4, 11.1). The high enzyme activity observed suggests that this aminopeptidase has an important role in the production of free amino acids during germination.     

Synthesis and structure activity relationships of novel non-peptidic metallo-aminopeptidase inhibitors

Racemic derivatives of 3-amino-2-tetralone were synthesised and evaluated for their ability to inhibit metallo-aminopeptidase activities. New compounds substituted in position 2 by methyl ketone, substituted oximes or hydroxamic acids as well as heterocyclic derivatives were evaluated against representative members of zinc-dependent aminopeptidases: leucine aminopeptidase (E.C. 3.4.11.1), aminopeptidase-N (E.C. 3.4.11.2), Aeromonas proteolytica aminopeptidase (E.C. 3.4.11.10), and the aminopeptidase activity of leukotriene A(4) hydrolase (E.C. 3.3.2.6). Several compounds showed K(i) values in the low micromolar range against the 'one-zinc' aminopeptidases, while most of them were rather poor inhibitors of the 'two-zinc' enzymes. This interesting selectivity profile may guide the design of new, specific inhibitors of target mammalian aminopeptidases with one active site zinc.     

Isolation of the leucine aminopeptidase gene from Aeromonas proteolytica. Evidence for an enzyme precursor.

The leucine aminopeptidase of Aeromonas proteolytica (EC 3.4.11.10) is a monomeric metalloenzyme having the capacity to bind two Zn2+ atoms in the active site. Structural information of this relatively small aminopeptidase that could illuminate the catalytic mechanism of the metal ions is lacking; hence, we have obtained sequences from the purified enzyme, cloned the corresponding gene, and expressed the recombinant protein in Escherichia coli. The deduced primary amino acid sequence of this secreted protease suggests a potential signal peptide at the NH2 terminus. Expression of the recombinant and native proteins in E. coli and in extracts of culture media of A. proteolytica indicates that the aminopeptidase is secreted as an active and thermosensitive 43-kDa protein that is rapidly transformed to thermostable forms of 30 and 32 kDa. Comparison of the deduced amino acid sequence of the A. proteolytica leucine aminopeptidase with other Zn(2+)-binding metalloenzymes failed to show homologies to the consensus binding sequence His-Glu-X-X-His for the metal ion.     

Amino-benzosuberone: a novel warhead for selective inhibition of human aminopeptidase-N/CD13

This paper describes the design and synthesis of compounds belonging to a novel class of highly selective mammalian CD13 inhibitors. Racemic homologues of 3-amino-2-tetralone 1 were synthesised and evaluated for their ability to selectively inhibit the membrane-bound, zinc-dependent aminopeptidase-N/CD13 (EC 3.4.11.2). Some of these novel non-peptidic compounds are potent, competitive inhibitors of the mammalian enzyme, with K(i) values in the low micromolar range in spite of their minimal size (MW <200 Da). Moreover, they show an interesting selectivity profile against representative members of the aminopeptidase family, that is leucine aminopeptidase (EC 3.4.11.1), Aeromonas proteolytica aminopeptidase (EC 3.4.11.10) and the aminopeptidase activity of leukotriene A4 hydrolase (EC 3.3.2.6). The amino-benzosuberone derivative 4 is the most promising compound in terms of potency, stability and selectivity. A hypothetical binding mode of 4 to the catalytic zinc and several conserved active site residues is proposed, based on the observed structure-activity relationships, structural insights from aminopeptidase-N homologues of known three-dimensional structure.     

Leucine aminopeptidase in extracts of swine muscle

1. Leucine aminopeptidase (EC 3.4.1.1) has been demonstrated in swine muscle at a level of activity one-fifth that of the swine kidney. 2. The enzyme has been purified 110-fold by precipitation with ammonium sulphate, heat treatment and chromatography on Sephadex G-100. 3. The enzyme is heat-stable, but is rapidly inactivated below pH7. It requires Mg(2+) or Mn(2+) for activity. The Michaelis constant for leucine amide with Mg(2+)-activated enzyme is 5.0x10(-3)m. 4. Muscle leucine aminopeptidase is very similar to the kidney enzyme.     

Purification and characterization of human placental aminopeptidase A

Human placental aminopeptidase A (AAP) was purified 3,900-fold from human placenta and characterized. The enzyme was solubilized from membrane fractions with Triton X-100, then subjected to trypsin digestion, zinc sulfate fractionation, chromatographies with DE-52, Sephacryl S-300, and hydroxylapatite, affinity chromatography with Bestatin-Sepharose 4B, and finally immunoaffinity chromatography with the antibody against microsomal leucine aminopeptidase (LAP). Aminopeptidase A was completely separated from leucine aminopeptidase by the immunoaffinity chromatography. The apparent relative molecular mass (Mr) of the enzyme was estimated to be 280,000 by gel filtration. The purified enzyme was most active at pH 7.1 with L-aspartyl-beta-naphthylamide (L-Asp-NA) as substrate; the Km value for this substrate was 4.0 mmol/l in the presence of Ca2+. Human placental aminopeptidase A was markedly activated by alkaline earth metals (Ca2+, Sr2+, Ba2+), but strongly inhibited by metal chelating agents such as EDTA and o-phenanthroline. The highest activity was observed with L-glutamyl-beta-naphthylamide, while only minimal hydrolysis was found with some neutral and basic amino acid beta-naphthylamides.     

The Caenorhabditis elegans orthologue of mammalian puromycin-sensitive aminopeptidase has roles in embryogenesis and reproduction

Mammals possess membrane-associated and cytosolic forms of the puromycin-sensitive aminopeptidase (PSA; EC 3.4.11.14). Increasing evidence suggests the membrane PSA is involved in neuromodulation within the central nervous system and in reproductive biology. The functional roles of the cytosolic PSA are less clear. The genome of the nematode Caenorhabditis elegans encodes an aminopeptidase, F49E8.3 (PAM-1), that is orthologous to PSA, and sequence analysis predicts it to be cytosolic. We have determined the spatio/temporal gene expression pattern of pam-1 by using the promoter region of F49E8.3 to control expression in the nematode of a second exon translational fusion of the aminopeptidase to green fluorescent protein. Cytosolic fluorescence was observed throughout development in the intestine and nerve cells of the head. Neuronal expression was also observed in the tail of adult males. Recombinant PAM-1, expressed and purified from Escherichia coli, hydrolyzed the N-terminal amino acid from peptide substrates. Favored substrates had positively charged or small neutral amino acids in the N-terminal position. Peptide hydrolysis was inhibited by the metal-chelating agent 1,10-phenanthroline and by the aminopeptidase inhibitors actinonin, amastatin, and leuhistin. However, the enzyme was approximately 100-fold less sensitive toward puromycin (IC50, 135 mum) than other PSA homologues. Following inactivation of the enzyme, aminopeptidase activity was recovered with Zn2+, Co2+, and Ni2+. Silencing expression of pam-1 by RNA interference resulted in 30% embryonic lethality. Surviving adult hermaphrodites deposited large numbers of oocytes throughout the self-fertile period. The overall brood size was, however, unaffected. We conclude that pam-1 encodes an aminopeptidase that clusters phylogenetically with the PSAs, despite attenuated sensitivity toward puromycin, and that it functions in embryo development and reproduction of the nematode.     

Purification of human liver cytosolic epoxide hydrolase and comparison to the microsomal enzyme

Epoxide hydrolase (EC 3.3.2.3) was purified to electrophoretic homogeneity from human liver cytosol by using hydrolytic activity toward trans-8-ethylstyrene 7,8-oxide (TESO) as an assay. The overall purification was 400-fold. The purified enzyme has an apparent monomeric molecular weight of 58 000, significantly greater than the 50 000 found for human (or rat) liver microsomal epoxide hydrolase or for another TESO-hydrolyzing enzyme also isolated from human liver cytosol. Purified cytosolic TESO hydrolase catalyzes the hydrolysis of cis-8-ethylstyrene 7,8-oxide 10 times more rapidly than does the microsomal enzyme, catalyzes the hydrolysis of TESO and trans-stilbene oxide as rapidly as the microsomal enzyme, but catalyzes the hydrolysis of styrene 7,8-oxide, p-nitrostyrene 7,8-oxide, and naphthalene 1,2-oxide much less effectively than does the microsomal enzyme. Purified cytosolic TESO hydrolase does not hydrolyze benzo[a]pyrene 4,5-oxide, a substrate for the microsomal enzyme. The activities of the purified enzymes can explain the specific activities observed with subcellular fractions. Anti-human liver microsomal epoxide hydrolase did not recognize cytosolic TESO hydrolase in purified form or in cytosol, as judged by double-diffusion immunoprecipitin analysis, precipitation of enzymatic activity, and immunoelectrophoretic techniques. Cytosolic TESO hydrolase and microsomal epoxide hydrolase were also distinguished by peptide mapping. The results provide evidence that physically different forms of epoxide hydrolase exist in different subcellular fractions and can have markedly different substrate specificities.     

Kinetic and biochemical properties of CTP:choline-phosphate cytidylyltransferase from the rat brain

In order to investigate the mechanisms involved in some brain disorders at the membrane level, we studied the kinetics and biochemical properties of brain CTP:choline-phosphate cytidylyltransferase (EC 2.7.7.15), the rate-limiting enzyme of the two-step biosynthesis of phosphatidylcholine. This enzyme catalyzes the biosynthesis of CDPcholine from choline phosphate and CTP. We found that its subcellular localization (mainly in microsomal and cytosolic fractions) was different from that of phosphatidylethanolamine N-methyltransferase (EC 2.1.1.17), the enzyme of the alternative pathway for phosphatidylcholine synthesis. CTP:choline-phosphate cytidylyltransferase showed a Km of 10 mM for CTP and 0.3 mM for choline phosphate and exhibited a random mechanism. CDPcholine, the reaction product, was a competitive inhibitor of choline phosphate and CTP utilization and had a Ki of 0.090 mM. Both particulate and soluble enzymes required Mg2+ and exhibited an optimal pH at about 7. Cytosolic activity was enhanced by addition of unsaturated fatty acids or phospholipids extracted from brain membranes. Such an enhancement was increased with the centrifugation time used for preparing the soluble enzyme.     

Aminopeptidase M from human liver. I. Solubilization, purification, and some properties of the enzyme

Aminopeptidase M [EC 3.4.11.2] was purified 772-fold to homogeneity from the microsomal fraction of human liver, with a yield of 18.9%, by a combination of solubilization with 0.5% Triton X-100 and then 1 M urea and chromatography on columns of DEAE-cellulose, hydroxylapatite, Butyl-Toyopearl, and Sephacryl S-300. The purified enzyme had a molecular weight of 140,000 by SDS-polyacrylamide gel electrophoresis and of 280,000 by gel filtration on a column of TSK gel 2000 SW. It was reconstituted into proteoliposomes with asolectin, showing its amphiphilic nature. The aminopeptidase M from liver was found to be efficiently inhibited by bile acids. The enzyme was almost completely inhibited by chenodeoxycholic acid and 70-90% inhibited by cholic acid at a concentration of 6 mM. The extent of inhibition by conjugated and unconjugated bile acids was in the order: unconjugated greater than glycoconjugated greater than tauroconjugated bile acid, independent of the nature of the substrates used. The inhibition by the various bile acids was totally reversible. Further, it was immunochemically revealed that a considerable amount of liver aminopeptidase M was released into the bile duct. The role of the aminopeptidase M on the bile canalicular membrane and of the enzyme released in the bile duct is discussed in relation to the effects of bile acids.     

Porcine liver aminopeptidase B. Substrate specificity and inhibition by amino acids

Porcine liver aminopeptidase B[EC 3.4.11.6] is highly specific for hydrolysis of beta-naphthylamides of basic L-amino acids; the Km values for L-arginine beta-naphthylamide and L-lysine beta-naphthylamide were 0.035 and 0.12 mM, respectively. The enzyme was inhibited by various alpha-amino acids. Among basic amino acids, L-homoarginine and L-arginine were the most potent inhibitors, L-lysine and L-norarginine (alpha-amino-gamma-guanidinobutyric acid) being less inhibitory. Hydrophobic amino acids also inhibited the enzyme competitively. This suggests that there is a hydrophobic region that binds the side chain of the substrates or inhibitors in the specificity site of the enzyme. Studies on the inhibitions by L-arginine derivatives showed that blocking of the alpha-carboxyl or the alpha-amino group reduced the inhibitory effect of L-arginine. Porcine liver aminopeptidase B was not inhibited by puromycin, whereas bestatin inhibited the enzyme competitively with a Ki value of 1.4 X 10(-8) M. This enzyme had no kinin-converting activity.     

Participation of liver aldehyde oxidase in reductive metabolism of hydroxamic acids to amides

The liver enzyme responsible for the reduction of aromatic and heterocyclic hydroxamic acids to the corresponding amides was investigated with salicylhydroxamic acid, benzohydroxamic acid, anthranilhydroxamic acid, and nicotinohydroxamic acid. Rabbit liver cytosol exhibited significant reductase activities toward the hydroxamic acids under anaerobic conditions when supplemented with an electron donor of aldehyde oxidase. Similarly, rabbit liver aldehyde oxidase reduced these compounds to amides in the presence of its own electron donor, indicating that the reductase activities observed in the liver cytosol are due mainly to the cytosolic molybdoflavin enzyme. Furthermore, a significant reduction of salicylhydroxamic acid and nicotinohydroxamic acid was also observed, when an electron donor of aldehyde oxidase was added, with liver cytosols from hamsters, guinea pigs, rats, and mice. The cytosolic reductase activities toward salicylhydroxamic acid were markedly inhibited by menadione, an inhibitor of aldehyde oxidase.     

Modulation of the mutagenicity of three dinitropyrene isomers in vitro by rat-liver S9, cytosolic, and microsomal fractions following chronic ethanol ingestion.

The effects of chronic ethanol feeding of rats on the ability of liver fractions to modulate the bacterial mutagenicity of three dinitropyrene isomers (1,3-, 1,6- and 1,8-DNP), which require bacterial enzymes but not an exogenous enzyme source for activation, were studied. The mutagenicity of the DNP isomers toward S. typhimurium TA98 and TA100 was attenuated in the presence of post-mitochondrial supernatants (S9) from both ethanol-fed and pair-fed rats albeit, that from the ethanol-fed group was more efficient in lowering the mutagenicity. The cytosolic fraction from ethanol-fed rats enhanced the mutagenicity of all of the DNP isomers in TA100. The most notable enhancement was with 1,3-DNP in which a more than 4-fold enhancement was obtained. Cytosol from pair-fed rats enhanced only the mutagenicity of 1,3-DNP, this by 2.9-fold. Cytosolic NADPH-nitroreductase activity from ethanol-treated rats toward 1,6-, 1,8- and 1,3-DNP was increased 2.8-, 1.7- and 1.3-fold, respectively over pair-fed controls. Cytosolic NADH-nitroreductase from ethanol-fed rats was increased with 1,3-DNP (1.7-fold) and 1,8-DNP (1.4-fold) as substrates, but not with 1,6-DNP. Microsomes decreased the mutagenicity of DNP similarly to S9, i.e., fractions from ethanol-fed rats were more efficient than those of pair-fed rats in deactivating all the DNP isomers. Per mg of protein, detoxification of DNP by S9 was more efficient than with microsomes, thus both cytosolic and microsomal enzymes are required for maximal detoxification. In summary, ethanol feeding modulates both the augmented cytosolic activation of DNP to mutagens and the deactivation of the direct-acting mutagenicity of DNP by microsomes. In combination, as is the case with S9, the microsomal detoxifying activity outcompetes the cytosolic activation.     

Chymotrypsin inhibitory conformation of dipeptides constructed by side chain–side chain hydrophobic interactions

A complete series of configurationally isomers (L -L , L -D , D -L AND D -D ) of a dipeptide Leu-Phe benzyl ester have been synthesized and assayed for chymotrypsin. In the conformational analysis by 400 MMz ¹H NMR, the L -D and D -L isomers, but not hte L -L and D -D isomers, showed fairly large up field shifts (0.2–0.4 ppm) of Leu-βCH₂ and γCH proton signals, indicating the presence of shielding effects from the benzene ring. In addition to distinct signal splitting of Phe-βCH₂, the NOE enhancement observed between Leu-δCH₃ and Phe-phenyl groups revealed that these groups are in close proximity. These data indicated that L -D and D -L isomers from a hydrophobic core between side chains of adjacent Leu and Phe residues. When the dipeptides were examined for inhibition of chymotrypsin using Ac-Try-OEt as a substrate, the L -L isomer showed no inhibition, itself becoming a substrate. However, the other three isomers inhibited chymotrypsin in a competitive manner, and the D -L isomer was strongest with K_i of 2.2 × 10_?5 M . It was found that the D -L isomer was only slowly hydrolysed but the L (or D )-D isomer was not. H-D -Phe-L -Leu-OBzl with the inverse sequence of H-D -Leu-L -Pre-OBzl inhibited chymotrypsin more strongly (K_i = 6.3 × 10^?6 M ). Since the free acid analogue of the D -L isomer exhibited no inhibition, the benzyl ester moiety itself was thought to be involved in the enzyme inhibition. It is assumed that in the inhibitory conformation the ester-benzyl group fits the S₁ site of chymotrypsin, while the side chain-side chain complexing hydrophobic core fits the S₂ site.     

Photoaffinity labeling of membrane-bound porcine aminopeptidase N

To investigate the possible role of aminopeptidase N (alpha-aminoacyl-peptide hydrolase (microsomal), EC 3.4.11.2) in the transport of amino acids from oligopeptides, the modified amino acids Phe(N3) and Phe(N3, I) and the tetrapeptides Phe(N3) or Phe(N3, I)-L-or-DAla-Gly-Gly have been synthesized. The azido-amino acids were radioactively labeled by tritium or 125I before their coupling with the tripeptides. Their utilization as photoaffinity labels for aminopeptidase N has been studied. The modification imposed at the N-terminal residue of the tetrapeptides has not impaired their hydrolysis by porcine aminopeptidase N (same kinetic parameters as unmodified peptides). In addition, evidence is presented for a specific and reversible interaction in the dark of the azido-derivatives at the substrate recognition site of the enzyme. Upon photolysis, irreversible inactivation of aminopeptidase N and covalent attachment of Phe(N3, I) have been demonstrated. Soluble and membrane-bound aminopeptidases are both labeled to the same extent indicating that the free azido-amino acid preferentially reacts with the external part of the enzyme. Although the linkage of the azido-derivative is not strictly restricted to the region of the active site, the values obtained strongly suggest that 1 mol probe has been covalently attached per mol monomer of inhibited aminopeptidase.     

L-Leucinthiol — A potent inhibitor of leucine aminopeptidase

L-leucinthiol (2-amino-4-methyl-1-pentanethiol) was designed as an inhibitor of leucine aminopeptidase by analogy with sulfhydryl inhibitors of other zinc-containing peptidases. It was synthesized from L-leucinol and shown to be a potent competitive inhibitor of the microsomal aminopeptidase from porcine kidney (K_i = 2.2 × 10^?8M). The results suggest that the mechanism of aminopeptidase may be similar to that of other metalloproteases.     

Inhibition of aminopeptidase M by alkyl D-cysteinates

Ethyl D-cysteinate is a potent competitive inhibitor (Ki = 3.5 x 10(-7) M) of aminopeptidase M. D-cysteine and ethyl L-cysteinate inhibit more than two orders of magnitude less effectively. Inhibition studies on several n-alkyl esters of D-cysteine reveal an optimum at the n-butyl ester (Ki = 1.8 x 10(-7) M). The results are consistent with the hypothesis that the thiol group coordinates to Zn+2 at the active site and the alkyl group occupies the hydrophobic binding site for the side chain of the amino-terminal residue of substrates. Cytosolic leucine aminopeptidase is not significantly inhibited by ethyl D-cysteinate.