The structure and function of acid proteases. VI. Effects of acid protease-specific inhibitors on the acid proteases from Aspergillus niger var. macrosporus.

1. The Type B acid protease from Aspergillus niger var. macrosporus was inactivated by reaction with diazoacetyl-DL-norleucine methyl ester (DAN), DL-1-diazo-3-tosylamido-2-heptanone (DTH), and L-1-diazo-3-tosylamido-4-phenyl-2-butanone (DTPB) in the presence of cupric ions. The reaction with DAN took place with 1:1 stoichiometry. The enzyme was also inactivated by reaction with 1, 2-epoxy-3-(p-nitrophenoxy)-propane (EPNP) with concomitant incorporation of approximately two EPNP molecules per molecule of protein. Moreover, these reactions of DAN and of EPNP were markedly inhibited by pepstatin. These results seem to indicate that, as in the case of porcine pepsin [EC 3.4.23.1] and related acid proteases, the enzyme has two essential carboxyl groups at the active site, one reactive with DAN and related diazo reagents in the presence of cupric ions and the other reactive with EPNP, and that pepstatin binds in the vicinity of these residues. 2. The Type A acid protease from the same mold, on the other hand, was found to be markedly less sensitive to these specific inhibitors. Under conditions where the Type B enzyme was completely inactivated by DAN and related diazo reagents, only partial inactivation of this enzyme occurred. The effect of prior mixing of DAN and cupric ions on the pH profile of inactivation was also different from that for the Type B enzyme. Moreover, the Type A enzyme was not inactivated by EPNP. These results thus indicate that the nature of the active site of the Type A enzyme is rather different from that of the Type B enzyme and hence that the Type A enzyme belongs to a different class of acid proteases from the Type B enzyme.     

The structure and function of acid proteases. IV. Inactivation of the acid protease from Mucor pusillus by acid protease-specific inhibitors.

Mucor pusillus acid protease was rapidly inactivated with 1 : 1 stoichiometry by reaction with diazoacetyl-DL-norleucine methyl ester (DAN) in the presence of cupric ions. Cupric ions were essential for this inactivation. The rate of inactivation was maximal at around pH 6 when the enzyme was mixed with DAN and cupric ions without prior mixing of the reagents, and at pH 5.3 when DAN and cupric ions were mixed and incubated before addition to the enzyme solution. In both cases, the rate of inactivation decreased as the pH was either increased or decreased. The amino acid composition of an acid hydrolysate of the DAN-Modified enzyme was indistinguishable from that of the native enzyme except for the incorporation of about one norleucine residue per molecule of protein. The enzyme was also inactivated by reaction with 1,2-epoxy-3-(p-nitrophenoxy)-propane (EPNP). At the stage of about 90% inactivation, 1.50 residues of EPNP were incorporated per molecule of protein and the rate of inactivation followed pseudo-first order kinetics. The optimal pH for the inactivation was pH 3.0 and the rate of inactivation decreased as the pH was either increased or decreased. Furthermore, the enzyme was strongly inhibited by pepstatin, and the reactions of DAN and of EPNP was also inhibited significantly by prior treatment of the enzyme with pepstatin. These results suggest that the enzyme may have two essential carboxyl groups at the active site, one reactive with DAN in the presence of cupric ions and the other with EPNP, and that pepstatin binds part of the active site to inhibit the reactions with DAN and EPNP as well as the enzyme activity.     

Proteolytic enzymes in green wheat-leaves III. Inactivation of acid proteinase II by diazoacetyl-DL-norleucine methyl ester and 1,2-epoxy-3-(p-nitrophenoxy)-propane

Acid proteinase II isolated from green wheat leaves in a purifiedform was rapidly inactivated at pH=5.5 to 6.0 by a 50-fold molarexcess of diazoacetyl-DL-norleucine methyl ester (DAN) in thepresence of cupric ions which were essential for inactivation.The acid proteinase was also inactivated by reaction with 1,2-epoxy-3-(p-nitrophenoxy)-propane(EPNP). The inactivation by EPNP was much slower than by DANand the half-life of the activity was 24 hr. (Received February 6, 1978; )     

Structural study on the active site of porcine pepsin and Rhizopus chinensis acid protease. Spin labeling with diazoketone reagents

To investigate the active site structures of porcine pepsin and Rhizopus chinensis acid protease (RAP), spin label techniques were applied for these enzymes. Comparison of spin labeled porcine pepsin and RAP suggested that the active site cleft of porcine pepsin was narrower at the top, but wider at the bottom than that of RAP. Addition of pepstatin restricted the motion of the labeled nitroxide radicals. Under alkaline conditions, the enzymes changed their conformation discontinuously and irreversibly to open the active site clefts and to lose the binding ability for pepstatin. The denaturation points of both the enzymes were determined to be pH 6.2.     

Purification and properties of extracellular carboxyl proteases of acid-tolerant bacteria, isolated from compost

Four strains of acid-tolerant and protein-using bacteria were isolated from compost. Two of them, Gram-negative strains MB8 and MB11, were identified as a new genus close to Stenotrophomonas species MB8 and Burkholderia species MB11, respectively. Both bacteria produced extracellular carboxyl proteases (CP) in acid-casein-starch medium. The enzymes, termed CP MB8 and CP MB11, purified through ion exchange and gel filtration chromatographies had molecular weights of 61,000 (CP MB8) and 36,000 (CP MB11) as estimated by SDS-PAGE, and showed optimum activities with hemoglobin as a substrate at pH 3.5 (CP MB8) and pH 3.7 (CP MB11) at 55 degrees C. Both of the enzymes were not inhibited by pepstatin, DAN, or EPNP. These results suggest that both enzymes are members of the pepstatin-insensitive carboxyl proteinase family (EC 3.4.23.33), except for a larger molecular weight of the CP MB8 enzyme.     

Molecular characteristics of pepsinogen and pepsin from duck glandular stomach

1. Two procedures were developed for the preparation of duck pepsinogen, an enzyme from the family of aspartic proteases (EC 3.4.23.1) and its zymogen. 2. The amino acid composition, sugar content and the partial N- and C-terminal sequences of both the enzyme and the zymogen were determined. These sequences are highly homologous with the terminal sequences of chicken pepsin(ogen). 3. Duck pepsinogen and pepsin are unlike other pepsin(ogen)s in being relatively stable in alkaline media: pepsinogen is inactivated at pH 12.1, pepsin at pH 9.6. 4. Duck pepsin is inhibited by diazoacetyl-D,L-norleucine methyl ester (DAN), 1,2-epoxy-3(p-nitrophe-noxy)propane (EPNP), pepstatin and a synthetic pepsin inhibitor Val-D-Leu-Pro-Phe-Phe-Val-D- Leu. The pH-optimum of duck pepsin determined in the presence of synthetic substrate is pH 4. 5. Duck pepsin has a marked milk-clotting activity whereas its proteolytic activity is lower than that of chicken pepsin. 6. The activation of duck pepsinogen is paralleled by two conformational changes. The activation half-life determined in the presence of a synthetic substrate at pH 2 and 14 degrees C is 20 sec.     

Pepsinogen C and pepsin C from gastric mucosa of Japanese monkey. Purification and characterization.

A new pepsinogen component, pepsinogen C, was purified from the gastric mucosa of Japanese monkey. The chromatographic behavior of this component on DE-32 cellulose was coincident with that of pepsinogen III-2 previously reported (1), and final purification was performed by large-scale polyacrylamide disc gel electrophoresis. The molecular weight was 35,000 as determined by gel filtration. The ratios of glutamic acid to aspartic acid and of leucine to isoleucine were higher than those of other Japanese monkey pepsinogens. The activated form, pepsin C, had a molecular weight of 27,000 and contained a large number of glutamic acid residues. The optimal pH for hemoglobin digestion was 3.0. Pepsin C could scarcely hydrolyze the synthetic substrate, N-acetyl-L-phenylalanyl-3, 5-diiodo-L-tyrosine (APDT). 1, 2-Epoxy-3-(p-nitrophenoxy)propane (EPNP), p-bromophenacyl bromide, and diazoacetyl-DL-norleucine methyl ester (DAN) inhibited pepsin C [EC 3.4.23.3] in the same way as pepsin III-3 of Japanese monkey. The susceptibility to pepstatin of pepsin C was lower than that of pepsin III-3, and 500 times more pepstatin was required for the same inhibitory effect. The classification and nomenclature of Japanese monkey pepsinogens and pepsins are discussed.     

Amino acid sequences around 1, 2-epoxy-3-(p-nitrophenoxy)propane-reactive residues in rhizopus chinensis acid protease: homology with pepsin and rennin.

Two different peptides containing an aspartyl residue reactive with 1, 2-epoxy-3-(p-nitrophenoxy)propane (EPNP) in the acid protease from Rhizopus chinensis were isolated from a peptic digest of the EPNP-modified enzyme. One of the peptides was sequenced as Asp-Thr-Gly-Ser-Asp. The amino acid sequence had very high homology with those around the EPNP-reactive aspartyl residues in rennin (chymosin) [EC 3.4.23.4] and pepsin [EC 3.4.23.1]. The other peptide contained no methionine residue and gave the sequence: Asp-Thr-Gly-Thr-Thr-Leu. The N-terminal aspartyl residue of each peptide was deduced to be the EPNP-reactive site.     

Purification and characterization of rat pepsinogens whose contents increase with developmental progress.

Two pepsinogens, the contents of which increase with developmental progress, were purified from the gastric mucosa of the adult rat by ammonium sulfate fractionation and chromatography on DEAE-cellulose and DEAE-Sepharose CL-6B columns. The purified zymogens, designated as pepsinogens I and II, were each shown to be homogeneous by polyacrylamide gel disc electrophoresis. Pepsinogen II had a greater electrophoretic mobility toward the anode at pH 8.0 than pepsinogen I. The molecular weights of both zymogens were estimated to be 38,000 by SDS-polyacrylamide gel electrophoresis. The activated enzymes, pepsins I and II, each had the same molecular weight of 32,000. The pH optima for both enzymes were found to be 2.0. The enzymes showed high stabilities at pH 8.0, while they lost their activities within 60 min at pH 10.0. The enzymes were inhibited by pepstatin and diazoacetyl-DL-norleucine methyl ester (DAN). The activities of the enzymes in hydrolyzing N-acetyl-L-phenylalanyl-3,5-diiodo-L-tyrosine (APDT) were about 1/8 of that of porcine pepsin. These results suggest that pepsins I and II are very similar.     

N-terminal amino acid sequences of acid proteases: acid proteases from Penicillium roqueforti and Rhizopus chinensis and alignment with penicillopepsin and mammalian proteases.

The amino-terminal sequence (33 residues) of the acid protease from Penicillium roqueforti has been determined with an automated sequencer. The amino-terminal sequence of Rhizopus pepsin (published by Sepulveda, P., Jackson, K. W. & Tang, J. (1975) Biochem. Biophys. Res. Commun. 63, 1106-1112) has been extended from 27 residues to 39 residues. Also, it was found that two forms of Rhizopus pepsin differ in position 15, where Rhizopus pepsin I has an isoleucine and Rhizopus pepsin II a valine residue. The new sequences have been aligned with the amino-terminal sequences of penicillopepsin (EC 3.4.23.7), pig pepsin (EC 3.4.23.1), calf chymosin (EC 3.4.23.4), human pepsin (EC 3.4.23.2), human gastricsin (EC 3.4.23.3), and cow pepsin (EC 3.4.23.1). Residues 31-35 (numbering based on pig pepsin, Tang, J., Sepulveda, P., Marciniszyn, Jr., J., Chen, K.S.C., Huang, W.-Y. , Tao, N., Liu, D. & Lanier, P. (1973) Proc. Natl. Acad. Sci. U.S.A. 70, 3437-3739) are identical in all enzymes. This section contains one of the two aspartic acids (Asp-32) implicated in the active site. The similarity of the sequences provides strong evidence for the homology of these acid proteases.     

Covalent modification and active site-directed inactivation of a low molecular weight phosphotyrosyl protein phosphatase.

Covalent modification experiments were conducted in order to identify active site residues of the 18-kDa cytoplasmic phosphotyrosyl protein phosphatases. The enzyme was inactivated by diethyl pyrocarbonate, phenylglyoxal, cyclohexanedione, iodoacetate, iodoacetamide, phenylarsine oxide, and certain epoxides in a manner consistent with the modification of active site residues. Phenylglyoxal and cyclohexanedione both bind to the active site in a rapid preequilibrium process and thus act as active site-directed inhibitors. The pH dependencies of the inactivation by iodoacetate and by iodoacetamide were examined in detail and compared with rate data for the alkylation of glutathione as a model compound. The enzyme inactivation data permitted the determination of pKa values of two reactive cysteines at or near the active site. Although phosphomycin is simply a competitive inhibitor of the enzyme, it was found that 1,2-epoxy-3-(p-nitrophenoxy)propane (EPNP) and (R)- and (S)-benzylglycidol act as irreversible covalent inactivators, consistent with the importance of a hydrophobic moiety on the substrate in controlling substrate specificity. EPNP exhibits characteristics of an active site-directed inactivator, with a preequilibrium binding constant somewhat smaller than that of phosphate ion. The pH dependencies of inactivation of EPNP and (S)-benzylglycidol are identical to that observed for iodoacetamide and similar to that for iodoacetate, suggesting that they modify similar groups. Sequencing of the tryptic digests of the EPNP-labeled enzyme indicates that Cys-62 and Cys-145 are labeled. Phenylarsine oxide acts as a very slow, tight-binding inhibitor of the enzyme. The results are interpreted in terms of an active site model that incorporates a histidine-cysteine ion pair, similar to that present in papain.     

Purification and Some Enzymatic Properties of Acid Protease A and B of Scytalidium lignicolum ATCC 24568

Three kinds of acid proteases were purified from the culture filtrate of Scytalidium lignicolum ATCC 24568. About 3 mg of A–1, 6 mg of A–2 and 60 mg of B were obtained from one liter of culture broth. These purified enzymes were monodisperse by physicochemical criteria such as ultracentrifugal analysis and disc electrophoresis.

A–1 and A–2 were very similar to each other on their enzymatic properties except the small difference of isoelectric point. A–1 and A–2 were active between pH 3.0~3.5 toward casein, and stable between pH 2.5 and 5.5 for 20 hr at 37°C. Both enzymes were strongly inhibited by NBS, but not by EDTA, DFP and sulfhydryl reagents.

B was most active at pH 2.0, and stable at pH values between 1.5 and 5.0. This enzyme was also inhibited by NBS and KMnO₄, but not by EDTA, DFP and sulfhydryl reagents.

The molecular weights and isoelectric points of A–1, A–2 and B were 43,000, pH 3.6; 43,000, pH 3.8 and 22,000, pH 3.2, respectively.

A–1 and A–2 were not inhibited by S–PI and synthetic pepsin inhibitor such as diazoacetyl-dl-norleucine methylester (DAN) and 1,2-epoxy-3-(p-nitrophenoxy)-propane (EPNP). B was inhibited by EPNP, but not by S–PI and DAN.     

Purification and Properties of Extracellular Carboxyl Proteases of Acid-tolerant Bacteria,Isolated from Compost

Four strains of acid-tolerant and protein-using bacteria were isolated from compost. Two of them, Gram-negative strains MB8 and MB11, were identified as a new genus close to Stenotrophomonas species MB8 and Burkholderia species MB11, respectively. Both bacteria produced extracellular carboxyl proteases (CP) in acid-casein-starch medium. The enzymes, termed CP MB8 and CP MB11, purified through ion exchange and gel filtration chromatographies had molecular weights of 61,000 (CP MB8) and 36,000 (CP MB11) as estimated by SDS-PAGE, and showed optimum activities with hemoglobin as a substrate at pH 3.5 (CP MB8) and pH 3.7 (CP MB11) at 55°C. Both of the enzymes were not inhibited by pepstatin, DAN, or EPNP. These results suggest that both enzymes are members of the pepstatin-insensitive carboxyl proteinase family (EC 3.4.23.33), except for a larger molecular weight of the CP MB8 enzyme.     

An active-site peptide from pepsin C

Porcine pepsin C is inactivated rapidly and irreversibly by diazoacetyl-dl-norleucine methyl ester in the presence of cupric ions at pH values above 4.5. The inactivation is specific in that complete inactivation accompanies the incorporation of 1mol of inhibitor residue/mol of enzyme and evidence has been obtained to suggest that the reaction occurs with an active site residue. The site of reaction is the beta-carboxyl group of an aspartic acid residue in the sequence Ile-Val-Asp-Thr. This sequence is identical with the active-site sequence in pepsin and the significance of this in terms of the different activities of the two enzymes is discussed.     

Novel peptide-based pepsin inhibitors containing an epoxide group

1,2-Epoxy-3-(p-nitrophenoxy)propane (EPNP) is known to inhibit pepsin A and other aspartic proteinases by reacting with the active site aspartic acid residue(s). However, the reaction is considerably slow in general, and therefore, it is desirable to develop similar reagents that are capable of inhibiting these enzymes more rapidly. In the present study, we synthesized a series of novel inhibitors which have a reactive epoxide group linked with peptide by a hydrazide bond, with a general structure: Iva-L-Val-L-Val-(L-AA)(n)-N2H2-ES-OEt (n = 0 approximately 2) (Iva, isovaleryl; AA, bulky hydrophobic or aromatic amino acid residue; ES, epoxysuccinyl). These inhibitors were shown to inhibit porcine pepsin A remarkably faster than EPNP.     

Comparative pepstatin inhibition studies on individual human pepsins and pepsinogens 1,3 and 5(gastricsin) and pig pepsin A

Human gastric juice contains 3 major proteolytic components (pepsins1,3 and 5 or gastricsin). Pepsin 1 is increased in peptic ulcer and it's properties are relatively poorly understood. Studies with pepstatin the highly specific aspartic-protease inhibitor have therefore been carried out on individual active and proenzymes to assess any enzymic similarities. Human pepsin 1 was inhibited with high affinity similar to pepsin 3, whereas pepsin 5(gastricsin) was at least 40 times less sensitive. Inhibition of human pepsinogens 1,3 and 5 and pig pepsinogen A showed similar trends to the active enzymes. Studies using Sephadex gel filtration showed that pepstatin does not bind to pepsinogens and inhibition arises from pepstatin binding the pepsins released upon activation. Pepstatin inhibition was shown to be relatively independent of pH between 1.5 and 3.8 although at higher pH inhibition was less effective. The evidence suggests that pepsin 1 is similar to pepsin 3 and pepstatin inhibits by a one to one molecular binding to the active site. The explanation for the reduced affinity of pepstatin to pepsin 5(gastricsin) needs further study by co-crystallisation X-ray analysis.     

Comparative Pepstatin Inhibition Studies on Individual Human Pepsins and Pepsinogens 1,3 and 5(gastricsin) and Pig Pepsin A

Human gastric juice contains 3 major proteolytic components (pepsins1,3 and 5 or gastricsin). Pepsin 1 is increased in peptic ulcer and it's properties are relatively poorly understood. Studies with pepstatin the highly specific aspartic-protease inhibitor have therefore been carried out on individual active and proenzymes to assess any enzymic similarities. Human pepsin 1 was inhibited with high affinity similar to pepsin 3, whereas pepsin 5(gastricsin) was at least 40 times less sensitive. Inhibition of human pepsinogens 1,3 and 5 and pig pepsinogen A showed similar trends to the active enzymes. Studies using Sephadex gel filtration showed that pepstatin does not bind to pepsinogens and inhibition arises from pepstatin binding the pepsins released upon activation. Pepstatin inhibition was shown to be relatively independent of pH between 1.5 and 3.8 although at higher pH inhibition was less effective. The evidence suggests that pepsin 1 is similar to pepsin 3 and pepstatin inhibits by a one to one molecular binding to the active site. The explanation for the reduced affinity of pepstatin to pepsin 5(gastricsin) needs further study by co-crystallisation X-ray analysis.     

Human renin inhibition by a diazoacyl reagent: Relationship of the enzyme to other proteinases

Human renin is inactivated by a diazoacyl compound (diazoacetylglycine ethyl ester; N₂CHCO-Gly-OEt) in the presence of Cu(II). The mechanism of the inactivation is presumably identical to that which has been determined for pepsin and several other proteinases: esterification of the β-carboxyl of an aspartic acid residue at the active site of the enzyme. Renin's inhibition by the diazoacyl reagent, its specificity toward a hydrophobic sequence, and its inhibition by pepstatin, all suggest a close relationship to the acid proteinases, especially pepsin and cathepsin D. However, renin, a neutral proteinase, would be better classified together with other diazoacyl-inhibited enzymes by active site rather than pH optimum. The term “aspartic proteinase” is suggested for this group of enzymes.     

Extracellular acid and alkaline proteases from Candida olea

Candida olea 148 secreted a single acid protease when cultured at acidic pH. In unbuffered medium, the culture pH eventually became alkaline and a single alkaline protease was produced. This was the only proteolytic enzyme produced when the organism was grown in buffered medium at alkaline pH. Both proteolytic enzymes were purified to homogeneity (as assessed by SDS-PAGE). The Mr of the acid protease was 30900, the isoelectric point 4.5; optimum activity against haemoglobin was at 42 degrees C and pH 3.3. This enzyme was inactivated at temperatures above 46 degrees C and was inhibited by either pepstatin and diazoacetyl-norleucine methyl ester but was insensitive to inhibition by either 1,2-epoxy-3-(p-nitrophenoxy)-propane or compounds known to inhibit serine, thiol or metallo proteases. The acid protease contained 11% carbohydrate. The alkaline protease had an Mr of 23400 and isoelectric point of 5.4. The activity of this enzyme using azocoll as substrate above 42 degrees C and was inhibited by phenylmethyl-sulphonyl fluoride and irreversible inactivated by EDTA. The enzyme was also partially inhibited by DTT but was insensitive to either pepstatin or p-chloromercuribenzoic acid.     

Reaction of serine proteases with substituted 3-alkoxy-4-chloroisocoumarins and 3-alkoxy-7-amino-4-chloroisocoumarins: new reactive mechanism-based inhibitors

The time-dependent inactivation of several serine proteases including human leukocyte elastase, cathepsin G, rat mast cell proteases I and II, and human skin chymase by a number of 3-alkoxy-4-chloroisocoumarins, 3-alkoxy-4-chloro-7-nitroisocoumarins, and 3-alkoxy-7-amino-4-chloroisocoumarins at pH 7.5 and the inactivation of several trypsin-like enzymes including human thrombin and factor XIIa by 7-amino-4-chloro-3-ethoxyisocoumarin and 4-chloro-3-ethoxyisocoumarin are reported. The 3-alkoxy substituent of the isocoumarin is likely interacting with the S1 subsite of the enzyme since the most reactive inhibitor for a particular enzyme had a 3-substituent complementary to the enzyme's primary substrate specificity site (S1). Inactivation of several enzymes including human leukocyte elastase by the 3-alkoxy-7-amino-4-chlorisocoumarins is irreversible, and less than 3% activity is regained upon extensive dialysis of the inactivated enzyme. Addition of hydroxylamine to enzymes inactivated by the 3-alkoxy-7-amino-4-chloroisocoumarins results in a slow (t1/2 greater than 6.7 h) and incomplete (32-57%) regain in enzymatic activity at pH 7.5. Inactivation by the 3-alkoxy-4-chloroisocoumarins and 3-alkoxy-4-chloro-7-nitroisocoumarins on the other hand is transient, and full enzyme activity is regained rapidly either upon standing, after dialysis, or upon the addition of buffered hydroxylamine. The rate of inactivation by the substituted isocoumarins is decreased when substrates or reversible inhibitors are present in the incubation mixture, which indicates active site involvement. The inactivation rates are dependent upon the pH of the reaction mixture, the isocoumarin ring system is opened concurrently with inactivation, and the reaction of 3-alkoxy-7-amino-4-chloroisocoumarins with porcine pancreatic elastase is shown to be stoichiometric. The results are consistent with a scheme where 3-alkoxy-7-amino-4-chloroisocoumarins react with the active site serine of a serine protease to give an acyl enzyme in which a reactive quinone imine methide can be released. Irreversible inactivation could then occur upon alkylation of an active site nucleophile (probably histidine-57) by the acyl quinone imine methide. The finding that hydroxylamine slowly catalyzes partial reactivation indicates that several inactivated enzyme species may exist. The 3-alkoxy-substituted 4-chloroisocoumarins and 4-chloro-7-nitroisocoumarins are simple acylating agents and do not give stable inactivated enzyme structures.(ABSTRACT TRUNCATED AT 400 WORDS)