Studies on Bacterial Protease

A crystalline alkaline protease was prepared from B. amylosacchariticus, which was isolated as a strain of saccharogenic α-amylase-producing Bacillus subtilis. The enzyme was most active at pH values between 10.3 and 10.7 towards casein and was stable at pH values from 6 to 11 on twenty hour incubation at 30°C. Calcium ions were effective to stabilize the enzyme especially at higher temperatures. The enzyme was markedly inactivated by DFP as well as protease inhibitor from potato and slightly by surface active agents, but not affected by sulfhydryl reagents and divalent metal ions except Hg⁺⁺ .Hemoglobin was the best substrate for the enzyme and more than 20% of the peptide bonds were hydrolyzed. Of numerous synthetic peptides tested, only the two compounds, and , were found to be hydrolyzed. A cyclic peptide, gramicidin S, was split by the enzyme only at the peptide bond of -l-valyl-l-ornithyl-. Methyl n-butyrate and tributyrin were also good substrates for the alkaline protease obtained here.     

On the Specificity of a Rennin-like Enzyme from Mucor pusillus

The specificity of a rennin-like enzyme from Mucor pusillus Lindt was determined using synthetic peptides and oxidized insulin B chain as substrates. The results indicate that the enzyme exhibits specificity against aromatic, bulky or hydrophobic amino acid residues at both sides of the splitting point. The susceptibility of peptide substrates increases with the increase of their molecular size, indicating the significance of secondary interaction for hydrolysis. Z-tetrapeptides such as (the arrows show the bond split) are found as efficient substrates for the enzyme. The main points of cleavage in oxidized insulin B chain are; Phe-Val (1–2), Ala-Leu (14–15), Leu-Tyr (15–16), Tyr-Leu (16–17), and Phe-Phe (24–25).

The specificity of the M. pusillus enzyme is almost identical with that of the rennin-like enzyme from Mucor miehei, and similar to those of usual acid proteinases possessing tryp- sinogen activating ability, except that the latter enzymes show specificity against basic amino acid residues at the carbonyl-side of the splitting point.     

Studies on Mold Proteases

The substrate specificity of the crystalline acid protease obtained from Rhizopus chinensis was determined using B-chain of oxidized beef insulin and numerous synthetic peptides, comparing with that of several acid proteases from various sources. The peptide bonds susceptible to the action of Rhiz. acid protease were found to be mainly those involving the amino group of bulky amino acids. The enzyme split the B-chain of oxidized insulin at twelve sites of the peptide linkages and a certain similarity in the specificity was observed among the three acid proteases, Rhiz. protease, rennin and pepsin, all of which were known to show potent milk clotting activities.     

Studies on Bacterial Protease

Substrate specificity of the crystalline neutral protease of B. amylosacchariticus was investigated using the B-chain of oxidized beef insulin as the substrate, and the results were compared with those of proteases obtained from other strains of Bacillus subtilis. The neutral protease split the B-chain at eleven sites of the peptide linkages, indicating the narrow specificity as compared with subtilopeptidase A, The results also indicated that the peptide bonds susceptible to the action of the neutral protease were mainly those involving amino group of hydrophobic amino acids and tyrosine, with a few exception. The enzyme showed potent activities in casein digestion at near neutrality and in milk clotting at pH 5.6, whereas it was completely inert on esters and keratin, and only slightly active toward elastin.     

A new acid carboxypeptidase,O-1, fromAspergillus oryzae

A new acid carboxypeptidase was purified fromAspergillus oryzae grown on solid bran culture medium. The purified enzyme was found to be homogeneous by disc gel electrophoresis at pH 9.4 and isoelectric focusing. The enzyme was termedA. oryzae acid carboxypeptidase O-1 with isoelectric point 4.08. The substrate specificity of the new enzyme was investigated with proangiotensin, angiotensin, and bradykinin. Even when the proline was present at the penultimate position of the peptide, the enzyme rapidly hydrolyzed the carboxyterminal Pro-X (X=amino acid) peptide bond. TheK _m andk _cat values for angiotension (–Pro⁷–Phe⁸) at pH 3.7 and 30°C were 0.2 mM and 1.7 sec^–1, respectively.     

News & Notes: Specificity of a Neutral Zn-Dependent Proteinase from Thermoactinomyces sacchari Toward the Oxidized Insulin B Chain

The proteolytic specificity of the neutral Zn-dependent proteinase from Thermoactinomyces sacchari was determined by analysis of the peptides obtained after incubation with the oxidized insulin B chain as a substrate. The enzyme is an endopeptidase with broad specificity. In total, 12 peptide bonds in the B chain of insulin were hydrolyzed. The major requirement is that a hydrophobic residue such as Leu, Val, or Phe should participate with the α-amino group in the bond to be cleaved. However, hydrolysis of bonds at the N-terminal side of His, Thr, and Gly was also observed. The peptide bond Leu 15–Tyr 16 in the oxidized insulin B chain, which is the major cleavage site for the alkaline microbial proteinases, is resistant to the attacks of the enzyme from Thermoactinomyces sacchari and other neutral proteinases. The proteolytic activity of the Zn-dependent proteinase from T. sacchari is different from those of other metalloendopeptidases from microorganisms. Received: 10 November 1999 / Accepted: 15 December 1999     

Carboxyl Proteinase from the Wood Deteriorating Basidiomycete Pycnoporus coccineus :Substrate Specificity with Oxidized Insulin Peptide B1 ~ B16 and B15 ~ B24, Angiotensin and Proangiotensin

The specificity of highly purified carboxyl proteinase from Pycnoporus coccineus (formerly designated Trametes sanguined) was investigated with oligopeptides at pH 2.7. Hydrolysis of oxidized insulin peptide Bl ~ B16 was observed at two peptide bonds (His¹⁰-Leu¹¹ and Ala¹⁴-Leu¹⁵) during 3-hr incubation. The enzyme did not hydrolyze oxidized insulin peptide B15 ~ B24. Hydrolysis of angiotensin (formerly designated angiotensin II) was observed at the Tyr⁴-Ile⁵ bond. Hydrolysis of proangiotensin (formerly designated angiotensin I) was also at the Tyr⁴-Ile⁵ bond. In conclusion, peptide bonds which have a hydrophobic amino acid in the P′₁ position (as defined by Schechter and Berger, Biochem. Biophys. Res. Commun., 27, 157 (1967)) are preferentially cleaved by the trypsinogen activating carboxyl proteinase of Pycnoporus coccineus.     

Energetics of peptide bond formation at elevated temperatures

Summary The free energies of formation of the peptide bond between carbobenzoxy-glycine and L-phenylalanine amide in aqueous solution at temperatures up to 60°C were calculated from experimentally determined equilibrium constants. The reaction was catalyzed by a thermophylic enzyme. The thermodynamic energy barrier to peptide bond formation was found to decrease with increasing temperature: the standard free energy of peptide bond formation did appear to become negative in the region of 60°C. The possible significance of these results for peptide bond formation under prebiotic conditions is discussed.     

Action and Specificity of a Streptomyces Alkalophilic Proteinase

Action of the crystalline alkalophilic proteinase of a Streptomyces sp. which was active at pH values around 13, was investigated. The enzyme attacked various keratinous proteins such as wool, hair, feather etc., dissolving them with hydrolysis degrees from 2 to 6%, and the degradation of several keratins was observed under a microscope and scanning electron-microscope. Specificity of the enzyme was also studied using oxidized insulin B-chain. The enzyme cleaved most easily the peptide linkages between -Ser · His-, -Leu · Val-, -Phe · Tyr- and -Lys · Ala-, though it split several other sites of oxidized insulin B-chain, too, indicating that the action pattern of the proteinase is distinguished from those of alkaline proteinases so far reported.     

Synthesis of (±)-Xanthoxins

The enzymatic properties of P2-2 enzyme were determined by using cells of M. radiodurans. The enzyme was: most active at 60°C incubation temperature, stable at 40°C in neutral buffer, and inactivated by heating at 80°C for 15min. Maximal lytic activity occurred at pH 8.5 in Tris-HCl buffer. The range of enzyme stability was between pH 5.5 and 8. Bivalent metal ions, p-chloromercuribenzoate and monoiodo acetate inhibited lytic activity. The molecular weight was estimated to be 16,000 daltons by gel filtration on Sephadex G-75. The enzymatic digestion of peptidoglycans from the cell walls of M. radiodurans and M. lysodeikticus liberated free amino groups, but neither reducing groups nor N-acetylhexosamine, indicating that the enzyme was an endopeptidase. From analysis of the N-terminal amino acids of the digests, it is suggested that the P2-2 enzyme cleaves the peptide bond at the carboxyl group of D-alanine in peptidoglycan.     

The rate-limiting step in the folding of the cis-Pro167Thr mutant of TEM-1 β-lactamase is the trans to cis isomerization of a non-proline peptide bond

The stability and kinetics of unfolding and refolding of the P167T mutant of the TEM-1 β-lactamase have been investigated as a function of guanidine hydrochloride concentration. The activity of the mutant enzyme was not significantly modified, which strongly suggests that the Glu166–Thr167 peptide bond, like the Glu166–Pro167, is cis. The mutation, however, led to a significant decrease in the stability of the native state relative to both the thermodynamically stable intermediate and the fully unfolded state of the protein. In contrast to the two slower phases seen in the refolding of the wild-type enzyme, only one phase was detected in the refolding of the mutant, indicating a determining role of proline 167 in the kinetics of folding of the wild-type enzyme. The former phases are replaced by rapid refolding when the enzyme is unfolded for short periods of time, but the latter is independent of the time of unfolding. The monophasic refolding reaction of the mutant is proposed to reflect mainly the trans→cis isomerization of the Glu166–Thr167 peptide bond. © 1996 John Wiley & Sons, Inc.     

Murein hydrolase (N-acetyl-muramyl-l-alanine amidase) in human serum

An enzyme was identified in human serum which unlike lysozyme cleaved the amide bond between N-acetyl-muramic acid and l-alanine of the peptide side chain of the rigid layer (murein) of Escherichia coli. The N-acetylmuramyl-l-alanine amidase released all of the peptide side chains including those to which the lipoprotein is bound. A portion of the peptide side chains of the Micrococcus lysodeikticus murein was also hydrolysed from the polysaccharide chains. E. coli, M. lysodeikticus, Bacillus subtilis and Staphylococcus aureus were not killed by the amidase. Treatment of E. coli with EDTA or osmotic shock rendered the cells sensitive to the amidase and they were killed. Possible biological functions of the amidase are discussed.The enzyme was separated from lysozyme in human serum. Gel permeation chromatography indicated a molecular weight of the active enzyme of 82,000 while gel electrophoresis in the presence of sodium dodecyl sulfate revealed a molecular weight of 75,000. Thus, the enzyme probably consists of a single polypeptide chain. Incubation with neuraminidase rendered the amidase more basic suggesting the release of sialic acid residues. The modified glycoprotein disclosed an increased activity to murein. Enzyme activity was inhibited by p-chloromercuribenzene sulfonate and ethyleneglycol-bis(2-aminomethyl) tetraacetate (EGTA) at 1 and 0.2 mM concentration, respectively, whereas EDTA up to 5 mM was without effect. The amidase was also inactivated by agents that reduce disulfide bridges.     

1 '-(6-Hydroxyoctanoyl) nornicotine and 1 '-(7-Hydroxyoctanoyl) nornico-tine,Two New Alkaloids from Japanese Domestic Tobacco

A Streptomyces-pepsin inhibitor (S-PI or pepstatin Ac)-insensitive carboxyl proteinase was found in a still culture filtrate of Ganoderma lucidum (Mannen-take). The new carboxyl proteinase was purified, and about 15 mg of the purified enzyme was obtained from 15 liters of culture filtrate, with 13% recovery. The enzyme showed a single protein band on Polyacrylamide gel electrophoresis.

The enzyme was most active at pH 3.2 toward hemoglobin, and at pH 2.0 toward casein, and stable only in the narrow pH range of 3.5 to 5.2 even under mild treatment (37°C for 3hr). The molecular weight and isoelectric point were 36,000 and pH 5.3, respectively. The enzyme did not contain methionine.

The enzyme was characterized by the following points: (1) the proteolytic activity was not inhibited by carboxyl proteinase inhibitors such as S-PI, DAN, and EPNP; (2) the enzyme was very unstable; (3) the caseinolytic activity was very low compared with the hydrolysis of hemoglobin (about 15%); (4) the enzyme split preferentially the Phe(24)–Phe(25) bond of oxidized insulin B-chain at the rate of 50% for total hydrolysis. These characteristics were compared with the carboxyl proteinases isolated from Scytalidium lignicolum and Lentinus edodes, which were reported to be SPI- and DAN-insensitive carboxyl proteinases.     

Purification and Characterization of a Pepstatin-insensitive Carboxyl Proteinase from Polyporus tulipiferae (Irpex lacteus)

A pepstatin-insensitive carboxyl proteinase of Polyporus tulipiferae (formerly Irpex lacteus) was purified by methods including affinity chromatography with chymostatin as the ligand.

Although the enzyme’s maximum proteolytic activity on hemoglobin was at pH 2.8, it was not affected by carboxyl proteinase inhibitors such as DAN, EPNP, and pepstatin. On the other hand, the enzyme was inhibited by chymostatin competitively and its Ki value was estimated to be 1.6×10^?5 m. The enzyme was very heat labile and was inactivated completely at pH 4.6 by heating at 45°C for 15min. Polyporus enzyme and Scytalidium lignicolum enzyme A₁ hydrolyzed the same peptide bond of Z-tetrapeptides, but their primary specificities were slightly different. Polyporus enzyme as well as Ganoderma lucidum enzyme contains histidine, and the amino acid compositions of Polyporus enzyme and other pepstatin-insensitive carboxyl proteinases resembled each other.     

Limited proteolyses in pancreatic chymotrypsinogens and trypsinogens

In the 1950's, the specific cleavages of the peptide bonds occurring in bovine cationic chymotrypsinogen and trypsinogen were among the first examples of limited proteolyses. According to the split bond(s), the precursor is transformed into enzyme or different forms of zymogen or again into inert protein. The conversion of trypsinogen into trypsin triggers the activations of all the other enzyme precursors of pancreatic juice. In the pancreas, several factors oppose trypsinogen autoactivation, whereas, in the duodenum, all the conditions are favorable for trypsinogen activation by enteropeptidase.     

The slow step of folding of Staphylococcus aureus PC1 β-lactamase involves the collapse of a surface loop rate limited by the Trans to Cis isomerization of a non-proline peptide bond

We wished to test the hypothesis that the non proline cis to trans isomerization of the peptide bond at position 167 in the S. aureus β-lactamase PC1 exerts a significant controlling effect on the folding pathway of this enzyme. The previous data presented in support of this hypothesis could not rule out the effect of factors unrelated to non-proline cis/trans isomerization. We have used the plasmid pET9d to direct soluble overproduction of the S. aureus β-lactamase PC1 and a site-directed mutant (Ile 167 to Pro) in Escherichia coli. Following purification the proteins were subjected to a comparative analysis of the kinetics of unfolding and refolding using the techniques of near- and far-UV circular dichroism spectroscopy and fluorescence spectroscopy in conjunction with “double-jump” experiments. Results show that the fully-unfolded I167P mutant enzyme retains 20% of molecules in a fast-refolding form and that slower-refolding molecules fold faster than the recombinant wild-type enzyme. The final stage of folding involves folding of the Ω-loop into a conformation essential for enzymatic activity. In support of the original hypothesis, the folding of this Ω-loop is rate limited by the isomerization of the Glu 166-Ile 167 peptide bond. Proteins 33:550–557, 1998. © 1998 Wiley-Liss, Inc.     

Bovine pancreatic elastase II cleaves Gln-Ile bond

A peptidase (GICP) that cleaves the Gln-Ile bond of a peptide Gly-Ile-Asp-Val-Gln-Ile-Tyr(T-1), a sequence in phenylalanine oxidase, was purified from bovine pancreas. The purified enzyme had an Mr of approximately 29,000, as determined by SDS-PAGE, and its N-terminal sequence was identical to that of bovine pancreatic elastase II. The enzyme released Gly-Ile-Asp-Val-Gln and Ile-Tyr from T-1 (Km = 8.3 M k_cat = 2.1 s^–1) and the catalytic efficiency (2.6 × 10⁵ M^–1s^–1) was comparable to those of elastase II from porcine pancreas and rat mesenteric arterial bed perfusate. The P₁ site specificity of GICP toward oxidized insulin A and B chains suggested that major cleavage sites were the peptide bond at the C-terminal side of Gln, Leu, His, and Tyr residues.     

Crystal structure determination of a neutral neurotoxin BmK M4 fromButhus martensii Karsch at 0.20 nm

BmK M4 is a neutral neurotoxin in the BmK toxin series. It is medially toxic and belongs to group III cc-toxins. The purified sample was crystallized in rhombic space group P6 Using an X-ray diffraction technique, the crystal structure of BmK M4 was revealed by molecular replacement at 0.20 nm resolution. The model was refined. The final crystallographic R factor was 0.142 and the free R factor was 0.173. The root mean square deviation is 0.001 5 nm for the bond length and 1.753° for the bond angles. 64 water molecules were added to the asymmetric unit. The refined structure showed an unusual non-prolyl cis peptide bond at residue 10. The structure was compared with group II a-toxin BmK M8 (an acidic, weak toxin). The potential structural implications of the cis peptide bond were discussed.     

Polygalacturonic acid trans-eliminase of Xanthomonas campestris

Polygalacturonic acid trans-eliminase from the culture fluid of Xanthomonas campestris was purified 66-fold by acetone precipitation, citrate extraction and chromatography on diethylaminoethyl- and carboxymethyl-cellulose. The optimum pH is 9·5 in glycine–sodium hydroxide buffer. Up to 1mm-calcium chloride brings about a remarkable stimulation of the enzyme activity and, at this concentration, no other cations promote or inhibit enzyme action except Ba²⁺ ions, which cause complete inhibition. The enzyme degrades polygalacturonic acid in a random manner; it does not act upon polygalacturonate methyl glycoside, although it can cleave partially (68%) esterified pectin. The end products from polygalacturonic acid at 46% breakdown are unsaturated di- and tri-galacturonic acids, in addition to saturated mono-, di- and tri-galacturonic acids. Pentagalacturonic acid is split preferentially into saturated dimer plus unsaturated trimer, or into saturated trimer plus unsaturated dimer; at a lower rate, it is also split into monomer and unsaturated tetramer. Unsaturated pentamer is split into unsaturated dimer plus unsaturated trimer. Tetragalacturonic acid is split some-what preferentially at the central bond to form dimer and unsaturated dimer, but it is also split into monomer and unsaturated trimer. Unsaturated tetramer is split only at the central bond to yield only unsaturated dimer. Trigalacturonic acid is split into monomer and unsaturated dimer. Unsaturated trimer is cleaved into saturated dimer and probably 4-deoxy-l-5-threo-hexoseulose uronic acid, which has not yet been directly identified. Neither saturated nor unsaturated digalacturonic acid is attacked. The unsaturated digalacturonic acid was isolated and proved to be O-(4-deoxy-β-l-5-threo-hexopyranos-4-enyluronic acid)-(1→4)-d-galacturonic acid.     

Peptide syntheses mediated by Bacillus subtilis protease

Bacillus subtilis protease (Amano protease N) was examined as a catalyst for peptide bond formation via both the kinetically and thermodynamically controlled approaches. In general, the latter approach proved to be superior to the former, and a series of dipeptide syntheses and several segment condensations were achieved in good to high yields using the immobilized enzyme on Celite in acetonitrile with low water content.