Tatsushi Oka

Comparison of the specificities of various neutral proteinases from microorganisms

A comparative study was made of the specificities of six neutral proteinases of microorganisms which originated from Bacillus subtilis, Bacillus thermoproteolyticus, Pseudomonas aeruginosa, Streptomyces griseus, and Aspergillus oryzae against the oxidized insulin B-chain and various synthetic peptides. The major cleavages of the insulin molecule occurred at the peptide bonds of His-Leu, His-Leu, Ala-Leu, Tyr-Leu, 5 6 10 11 14 15 16 17 Gly-Phe, and Phe-Phe by all the enzymes. The experiment with synthetic peptides in 23 24 24 25 dicated that these enzymes preferentially hydrolyzed the peptide bonds containing amino groups of l-leucine and l-phenylalanine. The rates of activities against the both peptide bonds differed depending on the species of enzyme. An enhancement in the rate of hydrolysis was observed by the presence of an alanyl group as the amino acid whose carboxyl group formed the bond in the case of the P. aeruginosa enzyme, while the highest rate was seen by a tyrosyl group in the cases of the other enzymes. The peptidase activity of mold enzyme was considerably smaller in comparison with the bacterial enzymes. These results led to the consideration that neutral proteinases from microorganisms exhibit remarkably similar although not identical specificities irrespective of their origins.

Multiple proteolytic enzymes of Streptomyces fradiae. Production, isolation, and preliminary characterization.

Abstract Streptomyces fradiae exhibited the highest potency in the production of proteolytic enzymes among about 500 strains tested, all of Streptomyces genus. It was found that at least five proteinaess (peptide peptidohydrolases) and two peptidases such as leucine aminopeptidase and carboxypeptidase were produced by the organism, which were fractionable by column chromatography on carboxymethyl-cellulose. Various physicochemical and enzymic properties were studied. One of the enzymes that was easily crystallized showed similar characters to those of “keratinase”, previously discovered by Nickerson, Noval and Robison , and produced by the same organism in a keratin-salt medium. A further comparative study on the proteolytic enzyme system of S. fradiae cultured in keratin salt medium and that cultured in the ordinary non-keratinic medium disclosed no difference between them.

Subtilisin BPN′: Kinetic study with oligopeptides

Abstract The present study was undertaken to investigate the effects of neighboring residues surrounding the susceptible peptide bond on the appearance of specificity in subtilisin BPN′. For the purpose, a kinetic study was made using various synthetic oligopeptides, (A or B = various d - or l -amino acid residues; n = 0, 1, and 2; the arrow shows the bond split) as substrates. The hydrolysis was more or less affected by the three amino acid residues on the N-terminal side and by the two amino acid residues on the C-terminal side from the sensitive l -leucine residue, depending on the nature of A or B in those peptides. These effects on hydrolysis were related mainly to catalysis ( K cat ) and little to binding ( K m ). The hydrolysis of peptide substrates was also affected when the terminal α-amino group within four amino acid residues of the catalytic point on the N-terminal side, or a terminal α-carboxyl group within one amino acid residue on the C-terminal side were unblocked. The former effect was related mainly to binding, but the latter to catalysis. An amino-blocking residue Z also influenced the hydrolysis of peptide substrates when it blocked the amino group of the amino acid residue donating the susceptible carboxyl group. The effect of Z decreased with the distance from the catalytic point, i.e., by elongating the peptide chain on the N-terminal side from the catalytic point. This was related only slightly to binding, mainly to catalysis.

Achromobacter protease I-catalyzed conversion of porcine insulin into human insulin

Abstract We have established a procedure for converting porcine insulin into human insulin using a serine protease from Achromobacter lyticus M497-1 which shows unique specificity against lysine residues on the carboxyl side of the splitting point. Desalanine-(B30)-insulin (DAI) was prepared by digestion of porcine insulin with Achromobacter protease. The coupling between DAI and Thr-OBut was performed by the same enzyme at pH 6.5 with a large excess of the amine component (Thr-OBut) in the presence of high concentrations of organic co-solvents. The highest yield was 85% by 20 h reaction at 37°C. The synthesized [Thr-OBut-B30]-insulin was isolated, then deprotected with trifluoroacetic acid in the presence of anisole to obtain semisynthetic human insulin.

Subtilisin bpn inactivation by chloromethyl ketone derivatives of peptide substrates

Abstract Two chloromethyl ketone derivatives of peptide substrates, such as those of carbobenzoxy- l -alanyl- l -phenylalanine (I) and carbobenzoxy- l -alanyl-glycyl- l -phenylalanine (II) were synthesized. On incubation of each with subtilisin BPN′, a much higher rate of inactivation of the enzyme was observed than that found previously by Shaw and Ruscica with the chloromethyl ketone derived from carbobenzoxy- l -phenylalanine (III). Inactivation with II was especially fast, the rate being over 200 times greater than that with III. α-Chymotrypsin was also more sensitive to the reagent II than to III. The inactivation of subtilisin BPN′ by II was further studied: Inactivation was partly protected by the presence of hydrocinnamic acid, and molar stoichiometry was confirmed. The pH-velocity profile of the inactivation suggested that deprotonation of an active site residue in the enzyme molecule was essential for inactivation by the reagent just as deprotonation is essential for enzyme activity. Amino acid analysis of the inactivated enzyme indicated one histidine residue less in the acid hydrolyzate.

The compound active site of Bacillus subtilis neutral protease: Some properties of six subsites

Abstract To investigate the properties of each of the six subsites (S1-S3 and S1′-S3′) which constitute the compound active site of a neutral protease from Bacillus subtilis, a kinetic study was made using various synthetic peptides such as Z-A-(Gly)n-Leu-Ala and Z-Gly-Leu-(Gly)n-B(A or B = various d - and l -amino acid residues; n = 0, 1, and 2) as substrates. These peptides were all split at the peptide bond containing the amino group of l -leucine. Thus, in a kinetic study it was possible to examine the properties of each subsite when it was occupied by different synthetic peptides in which the kinds of A or B and the number n were varied in the peptide. The study indicated that these subsites all have both stereo- and sidechain-specificity, although the contribution of each subsite decreased as the distance from the catalytic site increased. The properties of these subsites differ qualitatively from each other: Subsite S1 is concerned only with binding, but subsite S1′ with both binding and catalysis, irrespective of the kind of amino acid residues. On the other hand, the other four subsites are concerned either with catalysis only or with both catalysis and binding, depending upon the kind of residues adjacent to the respective subsites. It was found further that both subsites S1 and S2′ produced a cooperative effect on the hydrolysis of a peptide bond recognized by subsite S1′. Thus, a very selective substrate Z-Phe-Leu-Ala was discovered, with a proteolytic coefficient about 1000 times higher than that of Z-Gly-Leu-NH2, which had previously been thought of as a specific substrate for the enzyme.

Comparative specificity of microbial acid proteinases for synthetic peptides. III. Relationship with their trypsinogen activating ability

Abstract The specificities of acid proteinases from Aspergillus niger , Aspergillus saitoi , Rhizopus chinensis , Mucor miehei , Rhodotorula glutinis , and Cladosporium sp. , and that of swine pepsin, were determined and compared with ability of the enzymes to activate trypsinogen. Various oligopeptides containing l -lysine, Z-Lys-X-Ala, Z-Lys-(Ala) m , Z-Lys-Leu-(Ala) 2 , and Z-(Ala) n -Lys-(Ala) 3 (X = various amino acid residues, m = 1–4, n = 1–2) were used as substrates. Of the enzymes which are able to activate trypsinogen, most split these peptides at the peptide bond formed by the carbonyl group of l -lysine. For the peptides to be susceptible to the enzymes it was essential that the chain extended for two or three amino acid residues on the C-terminal side of the catalytic point, and that a bulky or hydrophobic amino acid residue formed the imino-side of the splitting point. The rate of hydrolysis was markedly accelerated by elongation of the peptide chain with l -alanine on the N-terminal side of the catalytic point. Thus, of the substrates used, Z-(Ala) 2 -Lys-(Ala) 3 was the most susceptible to the microbial acid proteinases possessing trypsinogen activating ability. On the other hand, M. miehei enzyme and pepsin, which do not activate trypsinogen, showed very little peptidase activity on the peptides.

Effect of secondary interaction on the enzymatic activity of subtilisin BPN′: Comparison with α-chymotrypsin, trypsin, and elastase

It is well-known that the enzymatic activity of certain proteinases is affected by amino acid residues distant from the catalytic point in a peptide substrate. The effect may be called “secondary interaction”, as has been proposed by Fruton [ 11. In y-chymotrypsin and subtilisin BPN’, the subsites which have been shown by X-ray studies to be involved in the binding of tripeptide chloromethyl ketone, Ser(214)-Trp(215)Gly(2 16) of the backbone chain of the former enzyme [2] and Ser( 125)-Leu( 126)-Cly( 127) in the latter [3], may possibly be related with their secondary interaction. This may be assumed to be true for the other serine proteinases, trypsin [4] and elastase. The effect of secondary interaction on the enzymatic activity of subtilisin BPN’ has previously been determined [5, 61 using Z-XqGly),-TyrTNH2 or Z-XqGly),-LeuTNH2 (X = various amino acid residues; m = 0, 1, and 2; the arrows show the bonds split) as substrates. The result indicated that hydrolysis is markedly increased by an increase in the peptide chain length from the catalytic point to the N-terminus in the substrate, where the specific amino acid residues Ltyrosine or L-leucine occupied the position at the carbonyl-side of the splitting point. A question then arises whether the effect of secondary interaction on the hydrolysis of peptide substrates is the same irrespective of the kind of amino acid residue at the carbonyl-side of the splitting point. The activity of the four serine proteinases, subtilisin BPN’, chymotrypsin, trypsin, and elastase is due to an identical catalytic apparatus (“charged relay system” with Asp-His-Ser); and though their specificities are

On the specificity of Bacillus subtilis neutral protease in relation to the compound active site

Abstract A previous paper indicated that a neutral protease of Bacillus subtilis has a large active site which can be divided into at least six subsites S 1 -S 3 and S 1 ′-S 3 ′, on both sides of the catalytic site. Each subsite accomodates one amino acid residue of a peptide substrate, and is numbered S 1 , S 2 , etc. towards the NH 2 -end and S 1 ′, S 2 ′, etc. toward the COOH-end. To investigate the correlation between these six subsites and the specificity of the enzyme, kinetic studies were made using various synthetic peptides as substrates. The results led to the conclusion that the specificity is not always determined by all the subsites. The three subsites S 1 , S 1 ′, and S 2 ′ were the ones mainly concerned with the development of specificity, while the others were not always implicated.

On the specificity of Pseudomonas aeruginosa alkaline proteinase with synthetic peptides

Abstract The specificity of Pseudomonas aeruginosa alkaline proteinase was studied using various synthetic peptides as substrates. Since it was found that the smallest peptide which is sensitive to the enzyme is a Z-tripeptide, in which an internal peptide bond is split, the primary specificity against the amino acid residue at either side of the splitting point was determined using various Z-tripeptides as substrates. The results indicated that the enzyme shows specificity against l -alanine at the imino side and l -phenylalanine at the carbonyl side of the splitting point, though its specificity is not as stringent as that seen with usual metal neutral proteinases. An experiment using l -alanine oligomers (from tetramer to hexamer) as substrates showed that the proteolytic activity increases drastically with increase in chain length, suggesting that the hydrolysis is markedly affected by amino acid residues more distant from the catalytic point in a peptide substrate (“secondary interaction”). The effect of secondary interaction was studied using various Z-tetra-or Z-pentapeptide substrates, which indicated that the specificity against peptides larger than Z-tripeptides was determined by secondary interaction, rather than by primary specificity, the presence of a hydrophobic or bulky residue being required at positions distant, rather than adjacent to the splitting point.