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Two enzymes were purified from the green alga Codium fragile, collected in 1988 at Fukuoka, Japan, by batch-wise ion-exchange extraction, affinity chromatography and ion-exchange high-performance liquid chromatography (HPLC) using Boc-Ala-Ala-Pro-Arg- pNA as a substrate. The molecular weights of the enzymes were estimated as 38 000 and 39 000, using gel filtration HPLC. The enzymes had the same optimal pH range of 7 to 9 for both activities, and an exclusively hydrolyzed peptide bond on the carboxyl-terminal side of the L-arginine of peptide p-nitroanilides. The ratios of the enzymatic activity for X-Arg- pNA to X-Lys- pNA were larger than 100. The enzymes exhibited 30 times higher activity toward Boc-Ala-Ala-Pro-Arg- pNA when compared with trypsin. The activities were strongly inhibited by diisopropyl phosphofluoridate (DFP), and partially inhibited by phenylmethylsulfonyl fluoride (PMSF), benzamidine, leupeptin and antipain. The isolated enzymes were presumed to be trypsin-like serine protease from their primary substrate specificities and inactivation behavior. [ABSTRACT FROM AUTHOR]

Seeds of Wisteria floribunda contain several kinds of cysteine proteinase inhibitor (cystatin). We purified and characterized one of these inhibitors, named WCPI-3. The molecular weight of WCPI-3 was estimated to be 17,500 and 15,700 by gel filtration and SDS-PAGE, respectively. The isoelectric point was 5.7. WCPI-3 formed an equimolar complex with native papain and the dissociation constant was estimated to be 6.1 nM. Complex formation between WCPI-3 and Cys25-modified papain, such as S-carboxy-methylated or S-carbamoylmethylated papain, could not be observed by gel filtration or native PAGE analysis. A peptide fragment derived from WCPI-3 digested by Achromobacter proteinase (lysyl endopeptidase) had the amino acid sequence of VVAGVNYRFVLK. The VVAG sequence in this fragment corresponds to the conserved sequence QVVAG which is considered to be one of binding regions to cysteine proteinases. The amino acid sequence of the amino-terminal portion (34 residues) of WCPI-3 was highly homologous to that of oryzacystatin from rice seeds.

The phenylthiazolone (PTA) of p-guanidinophenylalanine (GPA) was synthesized and the susceptibility of this compound to trypsin and related enzymes was compared with that of the PTA of arginine (Arg). Both PTA-GPA and PTA-Arg were almost completely and rapidly hydrolyzed by trypsin and pronase. PTA-Arg was hydrolyzed rapidly by thrombin, whereas PTA-GPA was less susceptible to this enzyme. The rates of hydrolysis of the two PTAs by alpha-chymotrypsin and papain were fairly slow. The specificity constant (kcat/Km) for the hydrolysis of PTA-GPA by trypsin was about 4 times larger than that of PTA-Arg. Thus, PTA-GPA, as well as PTA-Arg, behaves as a specific internal thioester substrate for trypsin and is the best one in the series of PTA substrates examined so far. However, PTA derivatives of GPA and Arg were hydrolyzed with kcat/Km values smaller than those of N alpha-benzoyl ethyl esters of L-GPA and L-Arg by factors of 4 and 17, respectively.

Three trypsin inhibitors were isolated from tubers of taro (Colocasia antiquorum var. nymphaifolia?) and named taro trypsin inhibitors (TTI)-I, -II, and -III. The final preparations were homogeneous by polyacrylamide gel electrophoresis and sodium dodecyl sulfate (SDS) polyacrylamide gel electrophoresis. The three inhibitors showed strong and stoichiometric inhibition against bovine trypsin and the inhibitor constants (Ki) were estimated to be of the order of 10(-9) to 10(-10) M. In contrast, they had only a weak capacity to inhibit bovine alpha-chymotrypsin and did not inhibit subtilisins (BPN' and Carlsberg), porcine pepsin, or papain. Each inhibitor appeared to be a protein with a molecular weight of 40,000 which could be dissociated into two subunits, both of which had a molecular weight of 20,000. The inhibitors formed complexes with trypsin at molar ratios of 1:2, suggesting that each subunit of these inhibitors can react with the enzyme in a 1:1 molar ratio. The three inhibitors had similar amino acid compositions and none of them contained carbohydrate or free sulfhydryl group. The antitryptic activity of all three inhibitors was suppressed by treatment with 1,2-cyclohexanedione (CHD) but not with 2,4,6-trinitrobenzenesulfonate (TNBS), thus demonstrating each of the inhibitors to contain an arginyl residue at the reactive site.

Several trypsin inhibitors with different mobilities on polyacrylamide gel electrophoresis occur in the tubers of taro (Colocasia antiquorum), and they each have a dimeric molecular weight of 40,000. Of all the constituent subunits, molecular weight 20,000, of the taro trypsin inhibitor (TTI), three major subunit components were separated by chromatography on SP-Sephadex C-25 in 8 M urea, and they were named protomers alpha, beta, and gamma in the order of their elution from the SP-Sephadex column. After removal or dilution of the urea, the three protomers could be either reassociated individually or hybridized with each other to form dimeric inhibitors. All of the reassociated dimers were powerful inhibitors of trypsin. Among them, each dimer derived from protomers alpha and gamma was a weak inhibitor of chymotrypsin, whereas the dimer of protomer beta did not inhibit the enzyme. Therefore TTI is presumed to be a mixture of heterogeneous and homogenous dimers whose properties reflect those of their constituent protomers. It was also proved that the major three trypsin inhibitors (TTI-I, TTI-II, and TTI-III) previously isolated from taro tubers are composed of protomers alpha and gamma, i.e., TTI-II is a heterogeneous dimer of protomers alpha and gamma, and TTI-I and TTI-III are homogeneous dimers of protomers alpha and gamma, respectively. The molecular weight of a trypsin-TTI complex saturated with trypsin was found to be 79,000, suggesting the formation of a tetrameric complex.

Ethyl N-benzoyl-p- and m-guanidino-DL-phenylglycinates (DL-Bz-p-GPG-OEt and DL-Bz-m-GPG-OEt), and ethyl N-benzoyl-p-guanidino-L- and D-phenylalaninates (L-Bz-p-GPA-OEt and D-Bz-p-GPA-OEt) were synthesized. The ester of the racemic p-guanidinophenylglycine derivative was completely hydrolyzed by trypsin, pronase, alpha-chymotrypsin, and thrombin, though hydrolysis by the latter two enzymes was much slower. Papain hydrolyzed this ester substrate stereospecifically at a moderate rate and left the ester derivative of the D-enantiomer unaltered. Optical resolution of DL-Bz-p-GPG-OEt with papain gave N-benzoyl-p-guanidino-L-phenylglycine (L-Bz-p-GPG-OH) and the ester of the D-enantiomer of this amino acid derivative. On the other hand, DL-Bz-m-GPG-OEt was completely hydrolyzed by pronase and was stereospecifically hydrolyzed by papain, but was unaffected by trypsin, alpha-chymotrypsin, and thrombin. The trypsin-catalyzed hydrolysis of N alpha-benzoyl-L-arginine p-nitroanilide (L-Bz-Arg-pNA) was inhibited competitively by this ester. The specificity constant (kcat/Km) for L-Bz-p-GPG-OEt was about 57 times smaller than that for a specific ester substrate, ethyl N alpha-benzoyl-L-argininate (LO-Bz-Arg-OEt), while the value for the D-enantiomer of the former is about 14 times larger than that for the D-enantiomer of the latter. L-Bz-p-GPA-OEt has a specificity constant comparable to that for L-Bz-Arg-OEt. The value for the former is about 51 times larger than that for L-Bz-p-GPG-OEt. This suggests that the existence of the beta-methylene group in L-Bz-p-GPA-OEt is important in relation to the higher susceptibility of the ester to trypsin-catalyzed hydrolysis. In contrast with the L-enantiomer, D-Bz-p-GPA-OEt was found to be as competitive inhibitor for the hydrolysis of L-Bz-Arg-pNA. A significant difference was found between the stereospecificities of hydrolysis of the ester substrates of the two amino acid derivatives by trypsin.

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.

A protease was purified 163-fold from Pronase, a commercial product from culture filtrate of Streptomyces griseus, by a series of column chromatographies on CM-Toyopearl (Fractogel), Sephadex G-50, hydroxyapatite, and Z-Gly-D-Phe-AH-Sepharose 4B using Boc-Ala-Ala-Pro-Glu-pNA as a substrate. The final preparation was homogeneous by polyacrylamide gel electrophoresis (PAGE), sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE), and gel isoelectric focusing. Studies on the substrate specificity with peptide p-nitroanilides revealed that this protease preferentially hydrolyzed peptide bonds on the carbonyl-terminal side of either glutamic acid or aspartic acid. It was most active at pH 8.8 for the hydrolysis of Boc-Ala-Ala-Pro-Glu-pNA. The molecular weight of the protease was estimated to be 20,000 by gel filtration on Sepharose 6B using 6 M guanidine hydrochloride as an eluent, and 22,000 by SDS-PAGE in the presence of 2-mercaptoethanol. The isoelectric point of the enzyme was 8.4. The enzyme was inactivated by diisopropyl phosphofluoridate (DFP) but not by p-chloromercuribenzoate (PCMB) or EDTA.

An aminopeptidase was purified about 4,000-fold from the clarified homogenate of bovine leukocytes by a series of column chromatographies on DEAE-cellulose, hydroxyapatite, Sephadex G-150, and DEAE-Toyopearl. The purified enzyme had a specific activity of 3.8 mumol X min-1 X mg-1 with arginine beta-naphthylamide (Arg-2-NNap) as substrate, and a minute amount of contaminating protein was found to be present by gel electrophoresis. The molecular weight of the enzyme was estimated to be 94,000 by gel filtration on Sephadex G-150. The enzyme had a broad substrate specificity and a pH optimum between 6.5 and 7.0 for the hydrolysis of alpha-aminoacyl beta-naphthylamides. It hydrolyzed beta-naphthylamides of basic, aliphatic, and aromatic amino acids, and also catalyzed the liberation of amino-terminal phenylalanine from phenylalanyl peptides. The enzyme was inhibited by bestatin, puromycin, 1,10-phenanthroline, sulfhydryl reagents, and a variety of heavy metal ions. Only the cobaltous ion stimulated the enzyme and the values of both Km and Vmax for Arg-2-NNap increased. In gross properties the present enzyme resembles porcine liver aminopeptidase reported previously (Kawata, S., et al. (1982) J. Biochem. 92, 1093-1101) very closely.

Porcine liver aminopeptidase was inactivated by various sulfhydryl-reactive reagents, whose inactivation rates were in the order: p-chloromercuribenzoate(PCMB) greater than HgCl2 greater than 2,2'-dithiodipyridine greater than 5,5'-dithiobis(2-nitrobenzoic acid)(DTNB). The processes of inactivation by these reagents did not follow pseudo-first-order kinetics, and prolonged incubation did not alter the level of maximum inactivation. The substrates provided no protection against the inactivation by DTNB, and the numbers of sulfhydryl groups titrated with the reagent were not influenced by the presence or absence of puromycin (a competitive inhibitor). The modification of sulfhydryl groups caused a slight increase in the Km value for the enzyme and a significant decrease of the Vmax value. There are two ionizable groups (pKe, 6.2; 7.8 and pKes, 6.0; 7.8) in the catalytic action of the enzyme. From the pKi vs. pH profile of inhibition with PCMB, the pK value of 7.8 does not correspond to the ionization of a sulfhydryl group. The thiol-modified enzyme was activated by cobalt ion, as was the native enzyme (Kawata, S., et al. (1982) J. Biochem. 92, 1093-1101). But in contrast with the native enzyme, the thiol-modified enzyme was activated about 2.5-fold and the maximum activation remained almost constant during prolonged incubation with cobalt ion. These results suggest that the sulfhydryl groups of the enzyme are located apart from the binding site of cobalt ion and do not participate directly in the catalytic process.

An aminopeptidase B from porcine liver was purified about 2,000-fold by ammonium sulfate fractionation and a series of chromatographies on hydroxyapatite, DEAE-cellulose, Sephadex G-150, hydroxyapatite and DEAE-Sepharose columns. The purified preparation was homogeneous on polyacrylamide gel electrophoresis. The molecular weight of the enzyme was about 58,000 as determined by gel filtration on Sephadex G-100 and disc gel electrophoresis in the presence of sodium dodecyl sulfate. The enzyme exhibited maximum activity at pH 7.5 for the hydrolysis of L-arginine beta-naphthylamide at 25 degrees C. The enzyme was labile to prolonged warming and freezing. The enzyme was markedly stimulated by chloride ion, and was inhibited by Cu2+, Zn2+, Cd2+, Hg2+, Pb2+, and metal chelating agents. p-Chloromercuribenzoate was an uncompetitive inhibitor of the enzyme with a Ki value of 1.8 X 10(-6) M. The enzyme was inhibited by 1,10-phenanthroline and the inhibition was of the mixed type with a K1 value of 1.9 X 10(-4) M but activity was restored by the addition of Zn2+, Co2+, and Fe2+.

An aminopeptidase was purified about 700-fold from porcine liver homogenate by ammonium sulfate fractionation and a series of column chromatographies on DEAE-cellulose, Sephadex G-150, DEAE-Sepharose, and hydroxyapatite. The purified enzyme had a specific activity of 4.6 mumol X min-1 X mg-1 using L-leucine beta-naphthylamide as the substrate. The molecular weight of the enzyme was about 96,000 as determined by Sephadex G-150 column chromatography. The enzyme had a broad specificity and a pH optimum between 6.5 and 7.0 for hydrolysis of alpha-aminoacyl-beta-naphthylamines, and it hydrolyzed the beta-naphthylamides of aliphatic, basic, and aromatic amino acids. The enzyme also hydrolyzed peptide substrates with phenylalanine residues as their amino-termini, but it did not hydrolyze L-phenylalanyl-L-proline. The enzyme was inhibited by metal-chelating agents, sulfhydryl reagents, heavy metals, bestatin, and puromycin. Activity of the enzyme inhibited by sulfhydryl-reactive reagents was restored by the addition of sulfhydryl compounds. The enzyme was activated by cobaltous ion and the values of both Km and Vmax increased. The activation was pH-dependent and above pH 7.5 cobalt ion behaved as an inhibitor of the enzyme. No metal ions other than cobaltous ion stimulated the enzyme appreciably.

The rates of hydrolysis of the ester, amide and anilide substrates of p-guanidino-L-phenylalanine (GPA) by Streptomyces griseus trypsin (S. griseus trypsin) were compared with those of arginine (Arg) substrates. The specificity constant (kcat/km) for the hydrolysis of GPA substrates by the enzyme was 2-3-times lower than that for arginine substrates. The kcat and Km values for the hydrolysis of N alpha-benzoyl-p-guanidino-L-phenylalanine ethyl ester (Bz-GPA-OEt) by S. griseus trypsin are in the same order of magnitude as those of N alpha-benzoyl-L-arginine ethyl ester (Bz-Arg-OEt), although both values for the former when hydrolyzed by bovine trypsin are higher by one order of magnitude than those for the latter. The specificity constant for the hydrolysis of Bz-GPA-OEt by S. griseus trypsin is much higher than that for N alpha-benzoyl-p-guanidino-L-phenylglycine ethyl ester (Bz-GPG-OEt). As with the kinetic behavior of bovine trypsin, low values in Km and kcat were observed for the hydrolysis of amide and anilide substrates of GPA by S. griseus trypsin compared with those of arginine substrates. The rates of hydrolysis of GPA and arginine substrates by S. griseus trypsin are about 2- to 62-times higher than those obtained by bovine trypsin. Substrate activation was observed with S. griseus trypsin in the hydrolysis of Bz-GPA-OEt as well as Bz-Arg-OEt, whereas substrate inhibition was observed in three kinds of N alpha-protected anilide substrates of GPA and arginine. In contrast, no activation by the amide substrate of GPA could be detected with this enzyme.

Phenylthiazolones (PTAs) of arginine and its homologs and analogs, homoarginine, norarginine (alpha-amino-gamma-guanidinobutyric acid), canavanine, and gamma-hydroxyarginine, were prepared. A steady-state kinetic analysis of the trypsin [EC 3.4.21.4]-catalyzed hydrolysis reactions was carried out and the kinetic parameters for these internal thioesters were compared with those for normal linear ester substrates. PTA-gamma-hydroxyarginine was so labile that hydrolysis by the enzyme could not be followed. PTA-arginine has a specificity constant (Kcat/Km) comparable to that for the Nalpha-unblocked arginine ester substrate, though the value is about 0.1% of that for a specific ester substrate, Nalpha-tosylarginine methyl ester. PTA derivatives of canavanine and homoarginine were hydrolyzed with Kcat/Km walues of the same order of magnitude as that for PTA-arginine. However, PTA-noraginine was much less susceptible to tryptic hydrolysis that PTA-homoarginine, while the linear esters of norarginine are known to be more susceptible than those of homoarginine.

An aminopeptidase was purified about 1,700-fold from seeds of Japanese apricot (Prunus mume Sieb.) by a seven-step procedure comprising extraction from seeds, ammonium sulfate fractionation, DEAE-cellulose chromatography, first and second DEAE-Sepharose chromatography, hydroxyapatite chromatography, and Sephadex G-200 gel filtration. The purified enzyme was shown to be homogeneous on polyacrylamide gel electrophoresis. The molecular weight of the enzyme was estimated to be about 56,000 by gel filtration on Sephadex G-200 and the isoelectric point was 4.9. The pH optimum for L-leucine beta-naphthylamide was between pH 6.5 and 7.0, and the enzyme was stable in the pH 5.0 to 8.3 region and up to 50 degrees C. The enzyme hydrolyzed a variety of aminopeptidase substrates with a free alpha-amino group. Of the amino acid beta-naphthylamides, the enzyme was highly specific for substrates with a hydrophobic side chain in the amino terminal residue. The enzyme was strongly inhibited by p-chloromercuribenzoate, heavy metals, diethyl pyrocarbonate, and photooxidation with methylene blue, but was not affected by thiol compounds, peptidase inhibitors of microbial origin, such as bestatin and puromycin, or metal chelating agents. No activation of the enzyme by metal ions was observed. These results suggest that the enzyme is a true aminopeptidase in which cysteine and histidine residues participate in the catalytic process, and is not classifiable as a metalloenzyme.

A proline iminopeptidase was purified about 18,000-fold from apricot seeds (Prunus armeniaca LINN.) by a five-step procedure comprised of extraction from seeds, ammonium sulfate fractionation, DEAE-cellulose chromatography, CM-Sepharose chromatography, and rechromatography on CM-Sepharose. The purified enzyme had a molecular weight of 220,000 by gel filtration and 55,000 by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. This indicates that the native enzyme may be composed of four identical subunits. The isoelectric point was 6.2 as determined by gel electrofocusing. The pH optimum for L-proline beta-naphthylamide was between pH 7.5 and 8.0, and the enzyme was stable in the pH 6.5-to-8.0 region and up to 40 degrees C. The enzyme was specific for L-proline beta-naphthylamide among various amino acid beta-naphthylamides, and it also hydrolzyed L-prolylglycine and L-prolylglycylglycine. The enzyme was strongly inhibited by p-chloromercuribenzoate, 5,5'-dithiobis(2-nitrobenzoic acid), N-ethylmaleimide, and heavy metal ions, but was not activated significantly by thiol compounds. Moreover, the enzyme was inactivated by diethyl pyrocarbonate, p-bromophenacyl bromide, and photooxidation, but was not affected by diisopropyl fluorophosphate, phenylmethanesulfonyl fluoride, bestatin, puromycin, or metal chelating agents. No activation of the enzyme was observed on addition of metal ions. These results suggest that the enzyme is not classifiable as a metalloenzyme, and that cysteine and histidine residues may participate in the enzyme activity.

A new chromogenic substrate, Na-benzoyl-p-guanidino-L-phenylalanine p-nitroanilide (Bz-GPA-pNA), was synthesized. This compound is a good substrate for bovine trypsin (Km = 1.56 X 10(-5) M, kcat = 0.081 s -1, at pH 8.2) and was hydrolyzed as fast as Na-benzoyl-L-arginine p-nitroanilide (Bz-Arg-pNA) with much the same kcat/Km values. But the values are two orders of magnitude smaller than those for ester substrates, Na-benzoyl-p-guanidino-L-phenylalanine ethyl ester (Bz-GPA-OEt) and Na-benzoyl-L-arginine ethyl ester (Bz-Arg-OEt). Substrate activation behavior was observed on tryptic hydrolysis of this new substrate in a substrate concentration range higher than about 5.0 X 10(-4) M in analogy with the trypsin-Bz-Arg-pNA system.

The rates of hydrolysis of N alpha-benzoyl-p-guanidino-L-phenylalaninamide (Bz-GPA-NH2) and N alpha-substituted p-nitroanilides (pNA) of GPA (benzyloxycarbonyl(Z)-GPA-pNA, benzoyl(Bz)-GPA-pNA and acetyl(Ac)-GPA-pNA) by bovine and porcine trypsins were compared with those of arginine (Arg) substrates. The amide type substrates of GPA were hydrolyzed as fast as those of Arg by the two enzymes with much the same kcat/Km values, though significant differences were found between the kcat and Km values of GPA derivatives and those of Arg derivatives. The kinetic behavior of porcine trypsin toward GPA substrates was almost the same as that of the bovine enzyme. The ratio of the kcat value for Bz-GPA-OEt to that for Bz-GPA-NH2 was much larger than that for the ester to amide substrates of arginine, suggesting that the conformational change of the active site of trypsin induced by a benzene ring in the side chain of Bz-GPA-OEt specifically increases the velocity of the deacylation process of the ester substrate. Remarkably low values of both kcat and Km were found for the tryptic hydrolysis of Z-GPA-pNA and Ac-GPA-pNA, as well as on that of Bz-GPA-pNA (Tsunematsu, H., et al. (1983) J. Biochem. 94, 123-128). Z-GPA-pNA is the best substrate for the two trypsins among the three N alpha-substituted anilide substrates of GPA.(ABSTRACT TRUNCATED AT 250 WORDS)

N alpha-Benzyloxycarbonyl-p-guanidino-L-phenylalanine beta-naphthylamide (Z-GPA-beta NA) was synthesized and the susceptibility of this compound to trypsin and related enzymes was compared with that of N alpha-benzyloxycarbonyl-L-arginine beta-naphthylamide (Z-Arg-beta NA). Both Z-GPA-beta NA and Z-Arg-beta NA were rapidly and almost completely hydrolyzed by trypsin and pronase. Z-Arg-beta NA was hydrolyzed slowly by thrombin, while Z-GPA-beta NA was not susceptible to this enzyme at all. The rate of hydrolysis of Z-GPA-beta NA by papain was slower than that of Z-Arg-beta NA. Neither beta-naphthylamide substrate was hydrolyzed by alpha-chymotrypsin. The specificity constant (kcat/Km) for the hydrolysis of Z-GPA-beta NA by trypsin was somewhat larger than that for the hydrolysis of Z-Arg-beta NA. Contributions of the benzene ring in the side chain of Z-GPA-beta NA to good binding of this substrate to the specificity site of this enzyme and to the poor fit of the scissile bond in the substrate molecule to the active serine residue are presumed from comparison of the individual kinetic parameters (Km and kcat) for the two beta-naphthylamide substrates. Z-GPA-beta NA was ascertained to be a useful substrate in the study of the binding and catalytic specificities of various trypsin-like enzymes.

In order to study the conformation of the side chain of lysine substrates bound to the active center of trypsin, two lysine analogs, cis- and trans-2,6-diamino-4-hexenoic acids (4,5-dehydrolysines) were synthesized and kinetic parameters for the hydrolysis of benzoyl methyl esters and phenylthiazolones of these analogs by this enzyme were compared with those of the corresponding lysine derivatives. The derivatives of cis-4,5-dehydrolysine were hydrolyzed much more slowly than those of lysine, owing largely to the small kcat values for the former. On the other hand, the derivatives of the trans-isomer were hydrolyzed at about the same rates as those of lysine and the values of both Km and kcat of the former are also similar to those of the latter. These results indicate that the conformation of the side chain of the lysine derivatives hydrolyzed by trypsin is such that the beta- and epsilon-carbons are in a trans-like conformation, as suggested by X-ray crystallographic studies of inhibitor-trypsin complex.

An enzyme bearing thrombin-like specificity has been purified to homogeneity from the venom of Trimeresurus flavoviridis (the Habu snake). The enzyme is a monomer with a molecular weight of 23,500 as determined by analytical gel filtration and sodium dodecyl sulfate polyacrylamide gel electrophoresis. The protein contains approximately 210 amino acid residues and has a relatively high content of aspartic acid and glutamic acid. The isoelectric point was 4.8 and the extinction coefficient at 280 nm for a 1% solution was 11.5. The enzyme acted directly on fibrinogen to form a fibrin clot with 2.0 NIH units. Analysis by high performance liquid chromatography of enzyme-treated fibrinogen revealed the release of a peptide identical in composition to thrombin-induced fibrinopeptide A, but no peptide corresponding to fibrinopeptide B was detected. The enzyme showed esterase and amidase activities on synthetic substrates containing arginine. The enzyme exhibited higher activity toward tosyl-L-arginine methyl ester (TAME) but 6-times lower activity toward benzoyl-L-arginine p-nitroanilide when compared with bovin thrombin. The esterase activity was inhibited by diisopropylfluorophosphate and at a slower rate by phenylmethanesulfonyl fluoride, but was least affected by tosyl-L-lysine chloromethyl ketone, showing that the enzyme is a serine protease like thrombin. The enzyme showed a bell-shaped pH dependence of kcat/Km for hydrolysis of TAME, with a maximum around pH 8.5.

A new enzyme which hydrolyzes anilide substrates of p-guanidino-L-phenylalanine in preference to those of arginine was found in the ascitic plasma from Ehrlich ascites tumor-bearing mice. The activity of this enzyme on N alpha-benzyloxycarbonyl-p-guanidino-L-phenylalanine p-nitroanilide was strongly inhibited by diisopropyl fluorophosphate and phenylmethanesulfonyl fluoride but not by sulfhydryl-reactive reagents and metal chelating agents. Peptide substrates containing p-guanidino-L-phenylalanine were hydrolyzed by this enzyme much faster than those containing arginine. These results suggest that this enzyme is a different type of serine protease from trypsin and thrombin. This enzyme was also found in the human gastric and colon cancer cells and their surrounding ascitic plasmas.