Mutational analysis of the autoprocessing site of subtilisin YaB-G124A

The potential residue at the autoprocessing site for improving processing efficiency was evaluated from hydrolysis of 19 cleavage-site-mimicking octapeptides, VTTXQTVP (-4 to +4), by the mature subtilisin YaB and YaB-G124A mutants. Both enzymes cleaved the octapeptides mainly at two sites, X-Q (A-site) and Q-T (B-site), at varied preferences. Based on the results above, Met(-1) of YaB-G124A was mutated and, as expected, extracellular enzyme production increased with Gln or Ala replacement, but decreased with Ile or Asp substitution. Together with previous structural studies, our results suggest that autoprocessing is dependent on not only the primary structure, but also the peptide flexibility around the processing site. Cleavage at the B-site resulted in a novel YaB mutant lacking the N-terminus Gln 1, which led the mutant enzyme to less enzymatic activity by 80% and less thermal stability by 20 degrees C, perhaps due to its ligation to the high-affinity calcium ion.     

Molecular cloning of the structural gene for alkaline elastase YaB, a new subtilisin produced by an alkalophilic Bacillus strain.

Alkaline elastase YaB is an extracellular serine protease of the alkalophilic Bacillus strain YaB. We cloned the structural gene, ale, and determined the nucleotide sequence. The mature enzyme (268 amino acids) was preceded by a putative signal sequence and a prosequence (27 and 83 amino acids, respectively). The mature enzyme was 55% homologous to subtilisin BPN'. Almost all the positively charged residues are predicted to be on the surface of the molecule, which would facilitate binding to elastin. The P1 substrate site-related sequences differed between alkaline elastase YaB and subtilisin BPN'.     

The specificity of macrophage elastase on the insulin B-chain.

The specificity of macrophage elastase obtained from mouse peritoneal exudative macrophages was determined in the hydrolysis of the oxidized insulin B-chain. This elastase hydrolysed two bonds, namely Ala-Leu and Tyr-Leu. The rate of hydrolysis of the latter was two to three times greater than that of the former. The hexapeptide Glu-Ala-Leu-Tyr-Leu-Val, obtained by cleavage of the insulin B-chain, was not hydrolysed by macrophage elastase. When EDTA was present, proteolysis of the B-chain was not observed. The macrophage elastase is therefore different from the neutrophil elastase in specificity and mechanism.     

Specificity of alkaline elastase Bacillus on the oxidized insulin A- and B-chains

The substrate specificity of alkaline elastase Bacillus from alkalophilic Bacillus sp. Ya-B was investigated using oxidized insulin A- and B-chains. Under time-limited cleavage, the initial cleavage site of the enzyme on the oxidized insulin A-chain and B-chain was at the leucine13-tyrosine14 bond and the leucine15-tyrosine16 bond, respectively. When the cleavage was completed, three major cleavage sites and three minor cleavage sites on the A-chain, and five major cleavage sites and four minor cleavage sites on the B-chain were found. However, most of the peptides produced after complete hydrolysis of the A- or B-chain by the enzyme were composed of four to six amino acid residues. The results suggest that this enzyme cleaves the oxidized insulin A- and B-chains in a block-cutting manner.     

Improved translational efficiency of subtilisin YaB gene with different initiation codons in Bacillus subtilis and alkalophilic Bacillus YaB

The ale gene specifying the subtilisin YaB produced by alkalophilic Bacillus YaB, has an unusual start codon UUG. Changing this codon to AUG and GUG increasedexpression of the ale gene in B. subtilis DB104 and in an ale deficient mutant strain YaB-DEC4. The relative translational efficiency order of the threeinitiation codons is AU G > GU G > UUG in B. subtilis DB104 and in YaB-DEC4. These data suggest that the preferred initiation codon is AUG for ale gene expression in Bacillus .     

Improved autoprocessing efficiency of mutant subtilisins E with altered specificity by engineering of the pro-region.

Modification of substrate specificity of an autoprocessing enzyme is accompanied by a risk of significant failure of self-cleavage of the pro-region essential for activation. Therefore, to enhance processing, we engineered the pro-region of mutant subtilisins E of Bacillus subtilis with altered substrate specificity. A high-activity mutant subtilisin E with Ile31Leu replacement (I31L) as well as the wild-type enzyme show poor recognition of acid residues as the P1 substrate. To increase the P1 substrate preference for acid residues, Glu156Gln and Gly166Lys/Arg substitutions were introduced into the I31L gene based upon a report on subtilisin BPN' [Wells et al. (1987) Proc. Natl. Acad. Sci. USA 84, 1219-1223]. The apparent P1 specificity of four mutants (E156Q/G166K, E156Q/G166R, G166K, and G166R) was extended to acid residues, but the halo-forming activity of Escherichia coli expressing the mutant genes on skim milk-containing plates was significantly decreased due to the lower autoprocessing efficiency. A marked increase in active enzyme production occurred when Tyr(-1) in the pro-region of these mutants was then replaced by Asp or Glu. Five mutants with Glu(-2)Ala/Val/Gly or Tyr(-1)Cys/Ser substitution showing enhanced halo-forming activity were further isolated by PCR random mutagenesis in the pro-region of the E156Q/G166K mutant. These results indicated that introduction of an optimum arrangement at the cleavage site in the pro-region is an effective method for obtaining a higher yield of active enzymes.     

Increased proteolytic resistance of ribonuclease A by protein engineering.

Although highly stable toward unfolding, native ribonuclease A is known to be cleaved by unspecific proteases in the flexible loop region near Ala20. With the aim to create a protease-resistant ribonuclease A, Ala20 was substituted for Pro by site-directed mutagenesis. The resulting mutant enzyme was nearly identical to the wild-type enzyme in the near-UV and far-UV circular dichroism spectra, in its activity to 2',3'-cCMP and in its thermodynamic stability. However, the proteolytic resistance to proteinase K and subtilisin Carlsberg was extremely increased. Pseudo-first-order rate constants of proteolysis, determined by densitometric analysis of the bands of intact protein in SDS-PAGE, decreased by two orders of magnitude. In contrast, the rate constant of proteolysis with elastase was similar to that of the wild-type enzyme. These differences can be explained by the analysis of the fragments occurring in proteolysis with elastase. Ser21-Ser22 was identified as the main primary cleavage site in the degradation of the mutant enzyme by elastase. Obviously, this bond is not cleavable by proteinase K or subtilisin Carlsberg. The results demonstrate the high potential of a single mutation in protein stabilization to proteolytic degradation.     

The effect of amino acid deletion in subtilisin E, based on structural comparison with a microbial alkaline elastase, on its substrate specificity and catalysis.

Subtilisin from a wide variety of Bacillus species has been extensively investigated as a promising target for protein engineering. In this study, we analyzed the substrate specificity of B. subtilis subtilisin E based on the structure of a new alkaline elastase produced by the alkalophilic Bacillus strain Ya-B, which has very high elastolytic activity. Despite the high homology of the primary sequences of both enzymes (54% identical), alkaline elastase was found to lack four consecutive amino acids which, in subtilisin, have been shown by X-ray analysis to lie close to the P1 binding cleft. To examine the influence of such a deletion in subtilisin on its substrate specificity, we constructed several mutants missing four amino acids by site-directed mutagenesis. When assayed with synthetic peptides, elastin and casein as substrates, a mutant lacking Ser161-Thr162-Ser163-Thr164 showed considerably lower specific activity toward the substrates for subtilisin, and its substrate specificity approached that of alkaline elastase. The results indicate that the deletion in subtilisin E influences the catalytic efficiency as well as the P1 specificity, and that this region is, in part, responsible for the difference in specificity between the two enzymes.     

The specificity of bovine spleen cathepsin S. A comparison with rat liver cathepsins L and B.

The peptide-bond-specificity of bovine spleen cathepsin S in the cleavage of the oxidized insulin B-chain and peptide methylcoumarylamide substrates was investigated and the results are compared with those obtained with rat liver cathepsins L and B. Major cleavage sites in the oxidized insulin B-chain generated by cathepsin S are the bonds Glu13-Ala14, Leu17-Val18 and Phe23-Tyr26; minor cleavage sites are the bonds Asn3-Gln4, Ser9-His10 and Leu15-Tyr16. The bond-specificity of this proteinase is in part similar to the specificities of cathepsin L and cathepsin N. Larger differences are discernible in the reaction with synthetic peptide substrates. Cathepsin S prefers smaller neutral amino acid residues in the subsites S2 and S3, whereas cathepsin L efficiently hydrolyses substrates with bulky hydrophobic residues in the P2 and P3 positions. The results obtained from inhibitor studies differ somewhat from those based on substrates. Z-Phe-Ala-CH2F (where Z- represents benzyloxycarbonyl-) is a very potent time-dependent inhibitor for cathepsin S, and inhibits this proteinase 30 times more efficiently than it does cathepsin L and about 300 times better than it does cathepsin B. By contrast, the peptidylmethanes Z-Val-Phe-CH3 and Z-Phe-Lys(Z)-CH3 inhibit competitively both cathepsin S and cathepsin L in the micromolar range.     

Nucleotide-induced changes in the proteolytically sensitive regions of myosin subfragment 1

Limited proteolytic digestions of myosin subfragment 1 (S-1) with elastase, subtilisin, papain, and thermolysin yield fragments that correspond within 1-2K daltons to the 25K, 50K, and 20K fragments produced by trypsin. While papain and thermolysin cut preferentially at the 26K/70K junction, elastase and subtilisin cleave both the 26K/70K and the 75K/22K junctions in S-1. Using the above proteases as conformational probes, we have previously demonstrated that the binding of actin is sensed at both the 26K/50K and the 50K/22K junctions [Applegate, D., & Reisler, E. (1983) Proc. Natl. Acad. Sci. U.S.A. 80, 7109-7112]. We report here that the binding of nucleotides at the active site is also sensed at both junctions. Both 2 mM MgADP and 5 mM MgATP slow the rate of elastase and subtilisin cleavage of the 95K heavy chain. With elastase, the 3-fold decrease in the rate of cleavage induced by nucleotides is evidenced at both the 26K/50K and the 50K/22K junctions. The analysis of subtilisin digestions is complicated by Mg nucleotide induced cleavage at a new site to produce a 91K fragment. Using N-methyl-6-anilinonaphthalene-2-sulfonyl chloride (MnsCl) to fluorescently label the 26K peptide, we demonstrate that the additional cleavage site is approximately 4K daltons from the N-terminal portion of the 95K heavy chain.(ABSTRACT TRUNCATED AT 250 WORDS)     

Purification of a new extracellular 90-kDa serine proteinase with isoelectric point of 3.9 from Bacillus subtilis (natto) and elucidation of its distinct mode of action.

A new extracellular 90-kDa serine proteinase with an isoelectric point (pI) of 3.9 was purified from Bicillus subtilis (natto). Microheterogeneity was detected in the 50-kDa protease of bacillopeptidase F with pI 4.4 reported previously by Wu et al. and the sequence for the first 25 amino acids in the internal region of the enzyme was analyzed: ATDGVEWNVDQIDAPKAWALGYDGA. The cleavage sites in the oxidized B-chain of insulin by the proteinase were CySO3H7-Gly8, Val12-Glu13, Try16-Leu17, and Phe25-Tyr26. The activity was inhibited by phenylmethylsulfonyl fluoride (PMSF) and chymostatin, while the activity was not inhibited by proteinaceous Streptomyces subtilisin inhibitor (SSI) or alpha 2-macroglubulin.     

Similarities and specificities of fungal keratinolytic proteases: comparison of keratinases of Paecilomyces marquandii and Doratomyces microsporus to some known proteases

Based on previous screening for keratinolytic nonpathogenic fungi, Paecilomyces marquandii and Doratomyces microsporus were selected for production of potent keratinases. The enzymes were purified and their main biochemical characteristics were determined (molecular masses, optimal temperature and pH for keratinolytic activity, N-terminal amino acid sequences). Studies of substrate specificity revealed that skin constituents, such as the stratum corneum, and appendages such as nail but not hair, feather, and wool were efficiently hydrolyzed by the P. marquandii keratinase and about 40% less by the D. microsporus keratinase. Hydrolysis of keratin could be increased by the presence of reducing agents. The catalytic properties of the keratinases were studied and compared to those of some known commercial proteases. The profile of the oxidized insulin B-chain digestion revealed that both keratinases, like proteinase K but not subtilisin, trypsin, or elastase, possess broad cleavage specificity with a preference for aromatic and nonpolar amino acid residues at the P-1 position. Kinetic studies were performed on a synthetic substrate, succinyl-Ala-Ala-Pro-Phe-p-nitroanilide. The keratinase of P. marquandii exhibited the lowest Km among microbial keratinases reported in the literature, and its catalytic efficiency was high in comparison to that of D. microsporus keratinase and proteinase K. All three keratinolytic enzymes, the keratinases of P. marquandii and D. microsporus as well as proteinase K, were significantly more active on keratin than subtilisin, trypsin, elastase, chymotrypsin, or collagenase.     

Construction and screening of a multi-point site-specific mutant library of subtilisin E with a set of oligonucleotides

A mutant library of subtilisin E containing random combinations of various mutagenized sites was constructed by one-round mutagenesis with 15 mutagenic oligonucleotides. Mutants were screened through dot blot hybridization and DNA sequencing. A single-point mutant (Met 222Ala) and a three-point (Asn 76Asp/Asn109Ser/ I le 205/Cys) mutant gene from the library were expressed. The mutant proteins exhibited conspicuously improved resistance to oxidation and heat treatment, as reported before. The results show that the library is reliable and very useful for protease subtilisin E engineering.     

Construction and screening of a multi-point site- specific mutant library of subtilisin E with a set of oligonucleotides

A mutant library of subtilisin E containing random combinations of various mutagenized sites wasconstructed by one-round mutagenesis with 15 mutagenic oligonucleotides. Mutants were screened through dot blot hybridization and DNA sequencing. A single-point mutant (Met 222Ala) and a three-point (Asn 76Asp/Asnl09Ser/ I le 205/Cys) mutant gene from the library were expressed. The mutant proteins exhibited conspicuously improved resistance to oxidation and heat treatment, as reported before. The results show that the library is reliable and very useful for protease subtilisin E engineering.     

Antiprotease targeting: altered specificity of alpha 1-antitrypsin by amino acid replacement at the reactive centre

Alpha-1 antitrypsin (alpha 1AT) is an efficient inhibitor of the human neutrophil proteases, elastase and cathepsin G. The reactive centre P1 residue (Met358) of alpha 1AT is important in defining the specificity of inhibition; furthermore, oxidation of this residue results in a loss of inhibitor activity. There is evidence that oxidative inactivation of alpha 1AT may be involved in the pathogenesis of pulmonary emphysema associated with cigarette smoking. We have studied the effect of a series of amino acid replacements at the active centre on the inhibition properties of alpha 1AT. The mutant proteins were produced in E. coli following in vitro mutagenesis of the alpha 1AT cDNA. Alpha-1-AT (Ile358), (Ala358) and (Val358) were efficient inhibitors of both neutrophil and pancreatic elastase, but not cathepsin G. Alpha-1-AT (Ala356, Val358) and alpha 1AT (Phe358) were specific for pancreatic elastase and cathepsin G respectively. Alpha-1-AT (Leu358) inhibited both neutrophil elastase and cathepsin G. These data show that, for effective inhibition, a potential cleavage site for the protease must be displayed at the alpha 1AT active centre. In each case, replacement of Met358 led to resistance to oxidative inactivation. Since alpha 1AT (Leu358) inhibits both neutrophil proteases and is resistant to oxidation, this variant may be of increased potential for the therapy of destructive lung disorders.     

Substrate specificity of Bacillus subtilis intracellular serine protease. Hydrolysis of insulin beta-chain, native ribonuclease A and p-nitroanilide peptide substrates

Intracellular serine protease, termed ISP-103, was isolated from Bacillus subtilis, strain 103. The substrate specificity of the enzyme was compared to that of secretory subtilisins. Similar to subtilisins, ISP-103 cleaves a single peptide bond Ala20-Ser21 within the native pancreatic ribonuclease A, which results in the accumulation of trypsin-sensitive ribonuclease S, consisting of a non-covalently bound S-peptide (20 amino acid residues) and S-protein (104 amino acid residues). The enzyme hydrolyzes a single peptide bond Leu15-Tyr16 of the B-chain of oxidized bovine insulin, in contrast to the subtilisins cleaving four additional bonds. ISP prefers Leu rather than Phe in the P1 binding site of the rho-nitroanilide peptide substrates and shows a more strict dependence of the activity on the presence of the hydrophobic residues in the P2 and P3 sites. The data obtained indicate that the substrate specificity of ISP, being within the borders of subtilisin specificity, is nevertheless much more restricted.     

Substrate specificity of carboxyl proteinase ofAspergillus sojae

The specificity and mode of action ofAspergillus sojae carboxyl proteinase I were investigated with the oxidized B-chain of insulin.A. sojae carboxyl proteinase I hydrolyzed primarily two peptide bonds in the oxidized B-chain of insulin, the Leu¹⁵-Tyr¹⁶ bond and the Phe²⁴-Phe²⁵ bond. Additional cleavage of the bond Tyr¹⁶-Leu¹⁷ was also noted.     

Thermal stable and oxidation-resistant variant of subtilisin E

A remarkable thermal stable and oxidation-resistant mutant was obtained using the random mutagenesis PCR technique on the mutant M222A gene of subtilisin E. Sequencing analysis revealed an A was replaced by G at nucleotide 671 of the subtilisin E gene, converting the asparagine codon (AAT) to serine codon (AGT) at position 118. The half-life of M222A/N118S enzyme activity, when heated at 65 degrees C, was approximately 80 min while the half-life of M222A and wild-type subtilisin E were 13 min and 15 min, respectively. This suggested the stability of the M222A/N118S mutant was five times greater than that of the wild-type enzyme. The mutant was also as oxidation resistant as the mutant M222A of subtilisin E. These results indicated the M222A/N118S mutant is both an oxidation-resistant and a heat-stable variant of subtilisin E.     

Redesigning the reactive site loop of the wheat subtilisin/chymotrypsin inhibitor (WSCI) by site-directed mutagenesis. A protein–protein interaction study by affinity chromatography and molecular modeling

A site-directed mutagenesis strategy was employed to obtain four mutants of wheat subtilisin/chymotrypsin inhibitor (WSCI), with the aim to produce inactive forms of this protein. The mutants were expressed in Escherichia coli as fusion proteins and, after the tag removal, were purified to homogeneity. Three mutants, containing a single mutation at the sequence positions 49 or 50, were named E49S, E49P and Y50G, respectively. These mutants exhibited anti-subtilisin activities comparable to that of the wild type protein; instead, anti-chymotrypsin activity was detectable only for the mutant E49S. A fourth mutant (M48P-E49G), containing a double amino acid substitution at the inhibitor reactive site (P1–P1′), was inactive against both subtilisin and chymotrypsin. In order to investigate the interactions between the putative susceptible enzymes and the mutated forms of WSCI, we performed time-course hydrolysis experiments by incubating samples of the mutants with subtilisin–agarose and chymotrypsin–agarose, respectively. These experiments yielded information on the E/I complex formation, as well as on the timing of the cleavage pattern of some of these mutants. Molecular modeling studies were carried out with the 3D models of the mutants and of their putative complexes with subtilisin and chymotrypsin. In terms of inter- and intra-chain H-bond networks, the observations made for each theoretical E/I complex were found to be fully coherent with experimental data (kinetic and time-course hydrolysis) and supplied specific modalities of interaction of each mutant with the enzyme counterpart.     

Degradation products of insulin generated by hepatocytes and by insulin protease

The enzymatic mechanisms for insulin breakdown by hepatocytes have not been established, nor have the degradation products been identified. Several lines of evidence have suggested that the enzyme insulin protease is involved in insulin degradation by hepatocytes. To identify the products of insulin generated by insulin protease and to compare them with those produced by hepatocytes, we have incubated insulin specifically iodinated at either the B-16 or the B-26 tyrosines with insulin protease and with isolated hepatocytes, separated the products on high performance liquid chromatography (HPLC), and identified the B-chain cleavages. Insulin-sized products were obtained by Sephadex G-50 filtration. These insulin-sized products were injected on reverse-phase HPLC, and the peaks of radioactivity were identified. The product patterns generated by the enzyme and by hepatocytes were essentially identical with both isomers. The products were also sulfitolized to prepare the S-sulfonate derivatives of the B-chain and B-chain peptides. Again, the patterns on HPLC generated by the enzyme and by hepatocytes with both isomers were identical. Each of the original product peaks was also sulfitolized and injected separately on HPLC to relate B-chain peptides with product peaks. Again, the peptide compositions of the product peaks for both enzyme and hepatocytes were essentially identical. To identify the cleavage sites in the B-chain of insulin produced by insulin protease, the peptides from the degradation of [125I]iodo(B-26)insulin were purified and submitted to automated Edman degradation to identify the cycle in which radioactivity appeared. Seven peptides with cleavages on the amino side of the B26 residue were identified, and the cleavage sites were determined. Cleavages were found between B-9 and B-10 (Ser-His), B-10 and B-11 (His-Leu), B-14 and B-15 (Ala-Leu), B-13 and B-14 (Glu-Ala), B-16 and B-17 (Tyr-Leu), B-24 and B-25 (Phe-Phe), and B-25 and B-26 (Phe-Tyr). Peptides were also isolated from [125I]iodoinsulin incubated with isolated hepatocytes, and the cleavage sites in several of these were determined. These agreed exactly with the cleavage sites identified generated by the enzyme. The major peptides generated by the degradation of [125I]iodo(B-16)insulin were also isolated and sequenced, again showing identical cleavage sites.(ABSTRACT TRUNCATED AT 400 WORDS)