Complete amino acid sequence of streptokinase and its homology with serine proteases

The complete amino acid sequence of streptokinase has been determined by automated Edman degradation of its cyanogen bromide and proteolytic fragments. The protein consists of 415 amino acid residues. Sequence microheterogeneity was found at two positions. The NH2-terminal 245 residues of streptokinase are homologous to the sequences of several serine proteases including bovine trypsin and Streptomyces griseus proteases A and B. The sequence alignment suggests that the active-site histidine-57 has changed to a glycine in streptokinase. The other active-site residues, aspartyl-102 and serine-195, are, however, present at the expected positions. Streptokinase also contains internal sequence homology between the NH2-terminal 173 residues and a COOH-terminal 162-residue region between residues 254 and 415. Moderate homology in predicted secondary structures also exists between these two regions. Although streptokinase is not a protease, these observations suggest that it has evolved from a serine protease by gene duplication and fusion. A COOH-terminal region of about 80 residues is apparently deleted from the second half of the duplicated structures. These observations further suggest that the three-dimensional structure of streptokinase likely contains two independently folded domains, each homologous to serine proteases.     

The nucleotide sequence of gene 21 of bacteriophage T4 coding for the prohead protease

We have sequenced gene 21 coding for the bacteriophage T4 prohead protease. The sequence codes for a protein of 212 amino acids (aa) with an Mr of 23,251. A second possible in-frame initiation site was also found which would code for an Mr 18,440 protein. Evidence is presented that this second site is used in vivo. The only striking homology of gp21 to other proteins is with the serine proteases. The protein is homologous to a short aa sequence around the active site, but has a His where the active site Ser is normally found. However, mutation of this His to Ser gave a functional protein that could not be inhibited by serine protease inhibitors. We have located three sites in the gene that give rise to temperature-sensitive mutations. One of these is towards the N-terminus of the gene, the other two flank the region that shows homology with serine proteases. Attempts to overproduce the protein in Escherichia coli failed due to the extreme lability of the enzyme. A frame-shift mutation in the gene was therefore constructed which allowed the synthesis of large amounts of a stable N-terminal fragment of the protein.     

The primary structure of the glutamic acid-specific protease of Streptomyces griseus

The amino acid sequence and part of the DNA sequence of a glutamic acid-specific serine protease from Streptomyces griseus is reported. This protease is shown to be homologous with other serine proteases. An improved purification protocol for this enzyme is described.     

Functional analysis of the intramolecular chaperone. Mutational hot spots in the subtilisin pro-peptide and a second-site suppressor mutation within the subtilisin molecule.

The N-terminal pro-peptide of 77 amino acid residues is essential for the folding of subtilisin, an alkaline serine protease from Bacillus subtilis. The synthetic pro-peptide has been shown to be capable of guiding the proper folding of denatured subtilisin to enzymatically active enzyme. Thus the pro-peptide serves as an intramolecular chaperone, which is removed by an autoprocessing reaction after the completion of the folding. With use of localized polymerase chain reaction random mutagenesis a total of 25 amino acid substitution mutations that affected subtilisin activities were isolated. These mutations occurred in a high frequency at the hydrophobic regions of the pro-peptide. For one of the mutations, M(-60)T, a second-site suppressor mutation, S(188)L, was isolated within the mature region. These results suggest that the pro-peptide consists of a few functional regions which interact with specific regions of the mature region of subtilisin during the folding process.     

The AprV5 Subtilase Is Required for the Optimal Processing of All Three Extracellular Serine Proteases from Dichelobacter nodosus

Dichelobacter nodosus is the principal causative agent of ovine footrot and its extracellular proteases are major virulence factors. Virulent isolates of D. nodosus secrete three subtilisin-like serine proteases: AprV2, AprV5 and BprV. These enzymes are each synthesized as precursor molecules that include a signal (pre-) peptide, a pro-peptide and a C-terminal extension, which are processed to produce the mature active forms. The function of the C-terminal regions of these proteases and the mechanism of protease processing and secretion are unknown. AprV5 contributes to most of the protease activity secreted by D. nodosus. To understand the role of the C-terminal extension of AprV5, we constructed a series of C-terminal-deletion mutants in D. nodosus by allelic exchange. The proteases present in the resultant mutants and their complemented derivatives were examined by protease zymogram analysis, western blotting and mass spectrometry. The results showed that the C-terminal region of AprV5 is required for the normal expression of protease activity, deletion of this region led to a delay in the processing of these enzymes. D. nodosus is an unusual bacterium in that it produces three closely related extracellular serine proteases. We have now shown that one of these enzymes, AprV5, is responsible for its own maturation, and for the optimal cleavage of AprV2 and BprV, to their mature active forms. These studies have increased our understanding of how this important pathogen processes these virulence-associated extracellular proteases and secretes them into its external environment.     

Primary structure of human pancreatic protease E determined by sequence analysis of the cloned mRNA

Although protease E was isolated from human pancreas over 10 years ago [Mallory, P. A., & Travis, J. (1975) Biochemistry 14, 722-729], its amino acid sequence and relationship to the elastases have not been established. We report the isolation of a cDNA clone for human pancreatic protease E and determination of the nucleic acid sequence coding for the protein. The deduced amino acid sequence contains all of the features common to serine proteases. The substrate binding region is highly homologous to those of porcine and rat elastases 1, explaining the similar specificity for alanine reported for protease E and these elastases. However, the amino acid sequence outside the substrate binding region is less than 50% conserved, and there is a striking difference in the overall net charge for protease E (6-) and elastases 1 (8+). These findings confirm that protease E is a new member of the serine protease family. We have attempted to identify amino acid residues important for the interaction between elastases and elastin by examining the amino acid sequence differences between elastases and protease E. In addition to the large number of surface charge changes which are outside the substrate binding region, there are several changes which might be crucial for elastolysis: Leu-73/Arg-73; Arg-217A/Ala-217A; Arg-65A/Gln-65A; and the presence of two new cysteine residues (Cys-98 and Cys-99B) which computer modeling studies predict could form a new disulfide bond, not previously observed for serine proteases. We also present evidence which suggests that human pancreas does not synthesize a basic, alanine-specific elastase similar to porcine elastase 1.     

Modeling of substrate and inhibitory complexes of histidine-aspartic protease

A three-dimensional structure of histo-aspartic protease (HAP), a pepsin-like enzyme from the causative agent of malaria Plasmodium falciparum, is suggested on the basis of homologous modeling followed by equilibration by the method of molecular dynamics. The presence of a His residue in the catalytic site instead of an Asp residue, which is characteristic of pepsin-like enzymes, and replacement of some other conserved residues in the active site make it possible for the enzyme to function by the covalent mechanism inherent in serine proteases. The detailed structures of HAP complexes with pepstatin, a noncovalent inhibitor of aspartic proteases, and phenylmethylsulfonyl fluoride, a covalent inhibitor of serine proteases, as well as with a pentapeptide substrate are discussed.     

Modeling of substrate and inhibitory complexes of histidine-aspartic protease

A three-dimensional structure of histo-aspartic protease (HAP), a pepsin-like enzyme from the causative agent of malaria Plasmodium falciparum, is suggested on the basis of homologous modeling followed by equilibration by the method of molecular dynamics. The presence of a His residue in the catalytic site instead of an Asp residue, which is characteristic of pepsin-like enzymes, and replacement of some other conserved residues in the active site make it possible for the enzyme to function by the covalent mechanism inherent in serine proteases. The detailed structures of HAP complexes with pepstatin, a noncovalent inhibitor of aspartic proteases, and phenylmethylsulfonyl fluoride, a covalent inhibitor of serine proteases, as well as with a pentapeptide substrate are discussed.     

One-step site-directed mutagenesis of the Kex2 protease oxyanion hole

BACKGROUND: Members of the subtilisin family of serine proteases usually have a conserved asparagine residue that stabilizes the oxyanion transition state of peptide-bond hydrolysis. Yeast Kex2 protease is a member of the subtilisin family that differs from the degradative subtilisin proteases in its high substrate specificity, it processes pro-alpha-factor, the precursor of the alpha-factor mating pheromone of yeast, and also removes the pro-peptide from its own precursor by an intramolecular cleavage reaction. Curiously, the mammalian protease PC2, a Kex2 homolog that is likely to be required for pro-insulin processing, has an aspartate in place of asparagine at the 'oxyanion hole'. RESULTS: We have tested the effect of making substitutions of the conserved oxyanion-hole asparagine (Asn 314) of the Kex2 protease. To do this, we have developed a rapid method of site-directed mutagenesis, involving homologous recombination of a polymerase chain reaction product in yeast. Using this method, we have substituted alanine or aspartate for Asn 314 in a form of Kex2 engineered for secretion. Transformants expressing the two mutant enzymes could be identified by failure either to produce mature alpha-factor or to mate. The Ala 314 enzyme was unstable but the Asp 314 enzyme accumulated to a high level, so that it could be purified and its activity towards various substrates tested in vitro. We found that, with three peptides that are good substrates of wild-type Kex2, the k(cal) of the Asp 314 enzyme was reduced approximately 4500-fold and its K(M) approximately 4-fold, relative to the wild-type enzyme. For the peptide substrate corresponding to the cleavage site of pro-alpha-factor, however, k(cat) of the Asp 314 enzyme was reduced only 125-fold, while the K(m) was increased 3-fold. Despite its reduced catalytic activity, however, processing of the mutant enzyme in vivo - by the intramolecular cleavage that removes its amino-terminal pro-domain - occurs at an unchanged rate. CONCLUSIONS: The effects of the Asn 314-Asp substitution reveal contributions to the reaction specificity of the Kex2 protease of substrate residues amino-terminal to the pair of basic residues at the cleavage site. Aspartate at the oxyanion hole appears to confer k(caf) discrimination between substrates by raising the energy barrier for productive substrate binding: this may have implications for pro-insulin processing by the PC2 protease, which has an aspartate at the equivalent position. The rate of intramolecular cleavage of pro-Kex2 may be limited by a step other than catalysis, presumably protein folding.     

Toward Fast Determination of Protein Stability Maps: Experimental and Theoretical Analysis of Mutants of a Nocardiopsis prasina Serine Protease

The stability of serine proteases is of major importance for their application in industrial processes. Here we study the determinants of the stability of a Nocardiopsis prasina serine protease using fast residual activity assays, a feature classification algorithm, and structure-based energy calculation algorithms for 121 micropurified mutant enzyme clones containing multiple point mutations. Using a multivariate regression analysis, we deconvolute the data for the mutant clones and find that mutations of residues Asn47 and Pro124 are deleterious to the stability of the enzyme. Both of these residues are situated in loops that are known to be important for the stability of the highly homologous α-lytic protease. Structure-based energy calculations with PEATSA give a good general agreement with the trend of experimentally measured values but also identify a number of clones that the algorithm fails to predict correctly. We discuss the significance of the results in relation to the structure and function of closely related proteases, comment on the optimal experimental design when performing high-throughput experiments for characterizing the determinants of protein stability, and discuss the performance of structure-based energy calculations with complex data sets such as the one presented here.     

Intracellular serine protease of Bacillus subtilis: sequence homology with extracellular subtilisins.

Intracellular serine protease was isolated from stationary-grown Bacillus subtilis A-50 cells and purified to homogeneity. The molecular weight of the enzyme is 31,000 +/- 1,000, with an isoelectric point of 4.3. Its amino acid composition is characteristically enriched in glutamic acid content, differing from that of extra-cellular subtilisins. The enzyme is completely inhibited with phenylmethylsulfonyl fluoride and ethylenediaminetetraacetic acid. Intracellular protease possesses negligible activity towards bovine serum albumin and hemoglobin, but has 5- to 20-fold higher specific activity against p-nitroanilides of benzyloxycarbonyl tripeptides than subtilisin BPN'. Esterolytic activity of the enzyme is also higher than that of subtilisin BPN'. The enzyme is sequence homologous with secretory subtilisins throughout 50 determined NH2-terminal residues, indicating the presence of duplicated structural genes for serine proteases in the B. subtilis genome. The occurrence of two homologous genes in the cell might accelerate the evolution of serine protease not only by the loosening of selective constrainst, but also by creation of sequence variants by means of intragenic recombination. Three molecular forms of intracellular protease were found, two of them with NH2-terminal glutamic acid and one minor form, three residues longer, with asparagine as NH2 terminus. These data indicate the possible presence of an enzyme precursor proteolytically modified during cell growth.     

Molecular cloning of cDNA for matriptase, a matrix-degrading serine protease with trypsin-like activity.

A major protease from human breast cancer cells was previously detected by gelatin zymography and proposed to play a role in breast cancer invasion and metastasis. To structurally characterize the enzyme, we isolated a cDNA encoding the protease. Analysis of the cDNA reveals three sequence motifs: a carboxyl-terminal region with similarity to the trypsin-like serine proteases, four tandem cysteine-rich repeats homologous to the low density lipoprotein receptor, and two copies of tandem repeats originally found in the complement subcomponents C1r and C1s. By comparison with other serine proteases, the active-site triad was identified as His-484, Asp-539, and Ser-633. The protease contains a characteristic Arg-Val-Val-Gly-Gly motif that may serve as a proteolytic activation site. The bottom of the substrate specificity pocket was identified to be Asp-627 by comparison with other trypsin-like serine proteases. In addition, this protease exhibits trypsin-like activity as defined by cleavage of synthetic substrates with Arg or Lys as the P1 site. Thus, the protease is a mosaic protein with broad spectrum cleavage activity and two potential regulatory modules. Given its ability to degrade extracellular matrix and its trypsin-like activity, the name matriptase is proposed for the protease.     

Models of the serine protease domain of the human antithrombotic plasma factor activated protein C and its zymogen.

Three-dimensional structural analysis of physiologically important serine proteases is useful in identifying functional features relevant to the expression of their activities and specificities. The human serine protease anticoagulant protein C is currently the object of many genetic site-directed mutagenesis studies. Analyzing relationships between its structure and function and between naturally occurring mutations and their corresponding clinical phenotypes would be greatly assisted by a 3-dimensional structure of the enzyme. To this end, molecular models of the protease domain of protein C have been produced using computational techniques based on known crystal structures of homologous enzymes and on protein C functional information. The resultant models corresponding to different stages along the processing pathway of protein C were analyzed for structural and electrostatic differences arising during the process of protein C maturation and activation. The most satisfactory models included a calcium ion bound to residues homologous to those that ligate calcium in the trypsin structure. Inspection of the surface features of the models allowed identification of residues putatively involved in specific functional interactions. In particular, analysis of the electrostatic potential surface of the model delineated a positively charged region likely to represent a novel substrate recognition exosite. To assist with future mutational studies, binding of an octapeptide representing a protein C cleavage site of its substrate factor Va to the enzyme's active site region was modeled and analyzed.     

Intramolecular chaperone: the role of the pro-peptide in protein folding.

Subtilisin, an alkaline serine protease, is produced in the bacterium as pre-pro-subtilisin; the pre-peptide of 29 amino acid residues is the signal peptide essential for the secretion of prosubtilisin from the cytoplasm into the culture medium. On the other hand, the pro-peptide of 77 residues covalently linked to the amino terminal end of the subtilisin intramolecularly guides the folding of subtilisin into the active enzyme. Importantly, the pro-peptide is not required for the enzymatic activity and is removed intramolecularly by autoprocessing upon the completion of the protein folding. In this review, I will first summarize all the data concerning the functions of the subtilisin pro-peptide. On the basis of these results, I shall discuss a new general concept, an intramolecular chaperone to explain the essential role of the pro-peptide in protein folding.     

Molecular cloning and nucleotide sequence of the 90k serine protease gene,hspK, from Bacillus subtilis (natto) No. 16

We previously reported purification and characterization of a 90k serine protease with pI 3.9 from Bacillus subtilis (natto) No. 16 [Kato et al. 1992 Biosci Biotechnol Biochem 56:1166]. The enzyme showed different and unique substrate specificity towards the oxidized B-chain of insulin from those of well-known bacterial serine proteases from Bacillus subtilisins. The structural gene, hspK, for the 90k serine protease was cloned and sequenced. The cloned DNA fragment contained a single open reading frame of 4302 bp coding a protein of 1433 amino acid residues. The deduced amino acid sequence of the 90k-protease indicated the presence of a typical signal sequence of the first 30 amino acids region and that there was a pro-sequence of 164 amino acid residues after the signal sequence. The mature region of the 90k-protease started from position 195 of amino acid residue, and the following peptide consisted of 1239 amino acid residues with a molecular weight of 133k. It might be a precursor protein of the 90k-protease, and the C-terminal region of 43k might be degraded to a mature protein from the precursor protein. The catalytic triad was thought to consist of Asp33, His81, and Ser259 from comparison of the amino acid sequence of the 90k-protease with those of the other bacterial serine proteases. The high-molecular-weight serine protease, the 90k-protease, may be an ancient form of bacterial serine proteases.     

The structure of rat mast cell protease II at 1.9-A resolution

The structure of rat mast cell protease II (RMCP II), a serine protease with chymotrypsin-like primary specificity, has been determined to a nominal resolution of 1.9 A by single isomorphous replacement, molecular replacement, and restrained crystallographic refinement to a final R-factor of 0.191. There are two independent molecules of RMCP II in the asymmetric unit of the crystal. The rms deviation from ideal bond lengths is 0.016 A and from ideal bond angles is 2.7 degrees. The overall structure of RMCP II is extremely similar to that of chymotrypsin, but the largest differences between the two structures are clustered around the active-site region in a manner which suggests that the unusual substrate specificity of RMCP II is due to these changes. Unlike chymotrypsin, RMCP II has a deep cleft around the active site. An insertion of three residues between residues 35 and 41 of chymotrypsin, combined with concerted changes in sequence and a deletion near residue 61, allows residues 35-41 of RMCP II to adopt a conformation not seen in any other serine protease. Additionally, the loss of the disulfide bridge between residues 191 and 220 of chymotrypsin leads to the formation of an additional substrate binding pocket that we propose to interact with the P3 side chain of bound substrate. RMCP II is a member of a homologous subclass of serine proteases that are expressed by mast cells, neutrophils, lymphocytes, and cytotoxic T-cells. Thus, the structure of RMCP II forms a basis for an explanation of the unusual properties of other members of this class.     

Indicain, a dimeric serine protease from Morus indica cv. K2

A high molecular mass serine protease has been purified to homogeneity from the latex of Morus indica cv. K2 by the combination of techniques of ammonium sulfate precipitation, hydrophobic interaction chromatography, and size-exclusion chromatography. The protein is a dimer with a molecular mass of 134.5 kDa and with two monomeric subunits of 67.2 kDa and 67.3 (MALDI-TOF), held by weak bonds susceptible to disruption on exposure to heat and very low pH. Isoelectric point of the enzyme is pH 4.8. The pH and temperature optima for caseinolytic activity were 8.5 and 80 degrees C, respectively. The extinction coefficient (epsilon280(1%)) of the enzyme was estimated as 41.24 and the molecular structure consists of 52 tryptophan, 198 tyrosine and 42 cysteine residues. The enzyme activity was inhibited by phenylmethylsulfonylflouride, chymostatin and mercuric chloride indicating the enzyme to be a serine protease. The enzyme is fairly stable and similar to subtilases in its stability toward pH, strong denaturants, temperature, and organic solvents. Polyclonal antibodies specific to enzyme and immunodiffusion studies reveal that the enzyme has unique antigenic determinants. The enzyme has activity towards broad range of substrates comparable to those of subtilisin like proteases. The N-terminal residues of indicain (T-T-N-S-W-D-F-I-G-F-P) exhibited considerable similarity to those of other known plant subtilases, especially with cucumisin, a well-characterized plant subtilase. This is the first report of purification and characterization of a subtilisin like dimeric serine protease from the latex of M. indica cv. K2. Owing to these unique properties the reported enzyme would find applications in food and pharma industry.     

A novel trypsin-like serine protease (hepsin) with a putative transmembrane domain expressed by human liver and hepatoma cells

Recombinant clones with cDNA inserts coding for a new serine protease (hepsin) have been isolated from cDNA libraries prepared from human liver and hepatoma cell line mRNA. The total length of the cDNA is approximately 1.8 kilobases and includes a 5' untranslated region, 1251 nucleotides coding for a protein of 417 amino acids, a 3' untranslated region, and a poly(A) tail. The amino acid sequence coded by the cDNA for hepsin shows a high degree of identity to pancreatic trypsin and other serine proteases present in plasma. It also exhibits features characteristic of zymogens to serine proteases in that it contains a cleavage site for protease activation and the highly conserved regions surrounding the His, Asp, and Ser residues that participate in enzyme catalysis. In addition, hepsin lacks a typical amino-terminal signal peptide. Hydropathy analysis of the protein sequence, however, revealed a very hydrophobic region of 27 amino acids starting 18 residues downstream from the apparent initiator Met. This region may serve as an internal signal sequence and a transmembrane domain. This putative transmembrane domain could be involved in anchoring hepsin to the cell membrane and orienting it in such a manner that its carboxyl terminus, containing the catalytic domain, is extracellular.     

Amino acid sequence of rat mast cell protease I (chymase)

The amino acid sequence has been determined for rat mast cell protease I (RMCP I), a product of peritoneal mast cells. The active enzyme contains 227 residues, including three corresponding to the catalytic triad characteristic of serine protease (His-57, Asp-102, and Ser-195 in chymotrypsin). A computer search for homology indicates 73% and 33% sequence identity of RMCP I with rat mast cell protease II from mucosal mast cells and bovine chymotrypsin A, respectively. When the structure of RMCP I is compared to those of cathepsin G from human neutrophils and two proteases expressed in activated lymphocytes, 48-49% of the sequences are identical in each case. RMCP I has six half-cystine residues at the same positions as in RMCP II, cathepsin G, and the two lymphocyte proteases, suggesting disulfide pairs identical with those reported for RMCP II. A disulfide bond near the active site seryl residue and substrate binding site, present in pancreatic and plasma serine proteases, is not found in RMCP I or in the other cellular proteases. These results indicate that RMCP I and other chymotrypsin-like proteases of granulocyte and lymphocyte origin are more closely related to each other than to the pancreatic or plasma serine proteases.