Molecular structure of Rarobacter faecitabidus protease I. A yeast-lytic serine protease having mannose-binding activity.

Rarobacter faecitabidus protease I (RPI) is a serine protease exhibiting lytic activity toward living yeast cells. RPI is similar to elastase in its substrate specificity and has a lectin-like affinity for mannose. The gene encoding RPI was cloned to elucidate its structure and function. And its nucleotide sequence revealed that it contains an open reading frame encoding a 525-amino acid protein. Homology comparison indicated that pre-pro-RPI consists of three domains: (1) an NH2-terminal prepro domain not found in the mature form of RPI, (2) a protease domain homologous to the trypsin family of serine proteases, and (3) a COOH-terminal domain homologous to the COOH-terminal part of Oerskovia xanthineolytica beta-1,3-glucanase and the NH2-terminal part of the ricin B chain, a lectin isolated from the part of the ricin B chain, a lectin isolated from the castor bean. The RPI gene and its mutant were subsequently expressed in Escherichia coli under its beta-galactosidase promoter to investigate the function of the COOH-terminal domain. The mutant RPI, whose COOH-terminal domain was truncated by site-directed mutagenesis, lost both its mannose-binding and yeast-lytic activity, although the protease activity was not affected. These findings suggest that the COOH-terminal domain actually participates in the mannose-binding activity and is required for yeast-lytic activity.     

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.     

Primary structure of two distinct rat pancreatic preproelastases determined by sequence analysis of the complete cloned messenger ribonucleic acid sequences

The mRNA sequences for two rat pancreatic elastolytic enzymes have been cloned by recombinant DNA technology and their nucleotide sequences determined. Rat elastase I mRNA is 1113 nucleotides in length, plus a poly(A) tail, and encodes a preproelastase of 266 amino acids. The amino acid sequence of the predicted active form of rat elastase I is 84% homologous to porcine elastase 1. Key amino acid residues involved in determining substrate specificity of porcine elastase 1 are retained in the rat enzyme. The activation peptide of the zymogen does not appear related to that of other mammalian pancreatic serine proteases. The mRNA for elastase I is localized in the rough endoplasmic reticulum of acinar cells, as expected for the site of synthesis of an exocrine secretory enzyme. Rat elastase II mRNA is 910 nucleotides in length, plus a poly(A) tail, and encodes a preproenzyme of 271 amino acids. The amino acid sequence is more closely related to porcine elastase 1 (58% sequence identity) than to the other pancreatic serine proteases (33-39% sequence identity). Predictions of substrate preference based upon key amino acid residues that define the substrate binding cleft are consistent with the broad specificity observed for mammalian pancreatic elastase 2. The activation peptide is similar to that of the chymotrypsinogens and retains an N-terminal cysteine available to form a disulfide link to an internal conserved cysteine residue.     

Purification and characterization of aqualysin I (a thermophilic alkaline serine protease) produced by Thermus aquaticus YT-1

Aqualysin I is an alkaline serine protease which is secreted into the culture medium by Thermus aquaticus YT-1. Aqualysin I was purified, and its apparent relative molecular mass was determined to be 28 500. The enzyme contained four Cys residues (probably as two cystines), and its amino acids composition was similar to those of cysteine-containing serine proteases (proteinase K, etc.) as well as those of subtilisins. The NH2-terminal sequence of aqualysin I showed homology with those of the microbial serine proteases. The optimum pH for the proteolytic activity of aqualysin I was around 10.0. Ca2+ stabilized the enzyme to heat treatment, and the maximum proteolytic activity was observed at 80 degrees C. Aqualysin I was stable to denaturing reagents (7 M urea, 6 M guanidine.HCl and 1% SDS) at 23 degrees C for 24 h. The enzyme hydrolyzed the ester bond of an alanine ester and succinyl-Ala-Ala-Ala p-nitroanilide, a synthetic substrate for mammalian elastase. The cleavage sites for aqualysin I in oxidized insulin B chain were not specific when it was digested completely.     

Purification and partial characterization of an elastolytic serine protease of Prevotella intermedia.

Elastolytic strains of Prevotella intermedia were isolated from pus samples of adult periodontal lesions. Elastase was found to associate with envelope, and it could be solubilized with guanidine-HCl. The enzyme was purified to homogeneity by sequential procedures including ion-exchange chromatography, gel filtration, and hydrophobic interaction chromatography. This elastase was a serine protease, and its mass was 31 kDa. It hydrolyzed elastin powder, but collagen and azodye-conjugated proteins were not degraded by this enzyme. Both synthetic substrates for human pancreatic (glutaryl-L-alanyl-L-alanyl-L-prolyl-L-leucine p-nitroanilide) and leukocyte elastase (methoxy succinyl-L-alanyl-alanyl-L-prolyl-L-valine p-nitroanilide) were hydrolyzed.     

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.     

Molecular cloning of complementary DNA encoding one of the human pancreatic protease E isozymes

We have cloned a DNA from a human pancreatic cDNA library using a cloned rat pancreatic elastase 1 cDNA as a probe, and determined its nucleotide sequence. This cDNA contains a coding region of 810 nucleotides which encodes a 270-amino-acid protein. The deduced amino acid sequence shows less than 60% homologies with rat and porcine pancreatic elastase 1, although its substrate binding region is homologous with those of the above elastases 1. When this deduced amino acid sequence was compared with known amino acid sequences of pancreatic proteases other than elastases, it was found to contain an amino acid sequence which was highly homologous with the N-terminal amino acid sequence of porcine pancreatic protease E. We also purified human pancreatic protease E isozymes from human pancreatic juice, and determined their N-terminal amino acid sequences. One of the isozymes does not hydrolyze elastin but does hydrolyze a synthetic substrate. Endoglycosidase F digests glycoside bonds of the isozyme. These results suggest that the cDNA cloned by us corresponded to one of the human protease E isozymes.     

Active site mapping of the serine proteases human leukocyte elastase, cathepsin G, porcine pancreatic elastase, rat mast cell proteases I and II. Bovine chymotrypsin A alpha, and Staphylococcus aureus protease V-8 using tripeptide thiobenzyl ester substrates

The primary subsite specificities of human leukocyte elastase, cathepsin G, porcine pancreatic elastase, rat mast cell proteases I and II, bovine chymotrypsin A alpha, and the protease from strain V-8 of Staphylococcus aureus have been mapped with a series of tripeptide thiobenzyl ester substrates of the general formula Boc-Ala-Ala-AA-SBzl, where AA represents one of 13 amino acids. In addition, the effects of a P2 Pro and P4 methoxysuccinyl and succinyl groups were investigated. In an attempt to introduce specificity and/or reactivity into the substrate Boc-Ala-Ala-Leu-SBzl(X), the 4-chloro-, 4-nitro-, and 4-methoxythiobenzyl ester derivatives were studied. Enzymatic hydrolyses of the substrates were measured in the presence of 4,4'-dithiobis(pyridine) or 5,5'-dithiobis(2-nitrobenzoic acid), which provided a highly sensitive assay method for free thiol. The thio esters were excellent substrates for the enzymes tested, and in many cases, the best substrates reported here have kcat/KM values higher than those reported previously. The best substrate for human leukocyte elastase was Boc-Ala-Pro-Nva-SBzl(Cl), which has a kcat/KM of 130 X 10(6) M-1 s-1. A very reactive rat mast cell protease substrate, Boc-Ala-Ala-Leu-SBzl(NO2), was also found. The S. aureus V-8 protease was the most specific enzyme tested since it hydrolyzed only Boc-Ala-Ala-Glu-SBzl. Substituents on the thiobenzyl ester moiety of Boc-Ala-Ala-Leu-SBzl resulted in decreased KM values with human leukocyte elastase and rat mast cell protease I when compared to the unsubstituted derivative.(ABSTRACT TRUNCATED AT 250 WORDS)     

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.     

Rat mast cell protease 4 is a beta-chymase with unusually stringent substrate recognition profile

Activated mast cells release a variety of potent inflammatory mediators including histamine, cytokines, proteoglycans, and serine proteases. The serine proteases belong to either the chymase (chymotrypsin-like substrate specificity) or tryptase (trypsin-like specificity) family. In this report we have investigated the substrate specificity of a recently identified mast cell protease, rat mast cell protease-4 (rMCP-4). Based on structural homology, rMCP-4 is predicted to belong to the chymase family, although rMCP-4 has previously not been characterized at the protein level. rMCP-4 was expressed with an N-terminal His tag followed by an enterokinase site substituting for the native activation peptide. The enterokinase-cleaved fusion protein was labeled by diisopropyl fluorophosphate, demonstrating that it is an active serine protease. Moreover, rMCP-4 hydrolyzed MeO-Suc-Arg-Ala-Tyr-pNA, thus verifying that this protease belongs to the chymase family. rMCP-4 bound to heparin, and the enzymatic activity toward MeO-Suc-Arg-Ala-Tyr-pNA was strongly enhanced in the presence of heparin. Detailed analysis of the substrate specificity was performed using peptide phage display technique. After six rounds of amplification a consensus sequence, Leu-Val-Trp-Phe-Arg-Gly, was obtained. The corresponding peptide was synthesized, and rMCP-4 was shown to cleave only the Phe-Arg bond in this peptide. This demonstrates that rMCP-4 displays a striking preference for bulky/aromatic amino acid residues in both the P1 and P2 positions.     

Substrate specificity of human pancreatic elastase 2

The substrate specificity of human pancreatic elastase 2 was investigated by using a series of peptide p-nitroanilides. The kinetic constants, kcat and Km, for the hydrolysis of these peptides revealed that this serine protease preferentially hydrolyzes peptides containing P1 amino acids which have medium to large hydrophobic side chains, except for those which are disubstituted on the first carbon of the side chain. Thus, human pancreatic elastase 2 appears to be similar in peptide bond specificity to the recently described porcine pancreatic elastase 2 [Gertler, A., Weiss, Y., & Burstein, Y. (1977) Biochemistry 16, 2709] but differs significantly in specificity from porcine elastase 1. The best substrates for human pancreatic elastase 2 were glutaryl-Ala-Ala-Pro-Leu-p nitroanilide and succinyl-Ala-Ala-Pro-Met-p-nitroanilide. However, there was little difference among substrates with leucine, methionine, phenylalanine, tyrosine, norvaline, or norleucine in the P1 position. Changes in the hydrolysis rate of peptides with differing P5 residues indicate that this enzyme has an extended binding site which interacts with at least five residues of peptide substrates. The overall catalytic efficiency of human pancreatic elastase 2 is significantly lower than that of porcine elastase 1 or bovine chymotrypsin with the compounds studied.     

Substrate specificity and inhibitors of a capillary injury-related protease from sheep lung lymph

A serine protease (Mr 70,000 to 75,000) appearing in sheep lung lymph after capillary damage induced by Escherichia coli endotoxin, oleic acid, or air emboli, was studied for its specificity toward a series of synthetic peptide and thioester substrates containing an Arg residue in the P1 position. High specificity constants (kcat/Km) were generally obtained with substrates having two or more basic amino acid residues, and with those having a Gln residues in the P2 position. Secondary enzyme-substrate interactions at sites more removed from the scissile bond are of importance, since a few peptides with two basic residues were hydrolyzed slowly, and the site of cleavage of natural peptides was influenced by the amino acid sequence beyond the immediate vicinity of the hydrolyzed bond. The properties of the enzyme and its pattern of specificity distinguish it from enzymes of the clotting cascade, from components of the complement system, and from lung and skin tryptase. The enzyme was inactivated by p-amidinophenylmethanesulfonyl fluoride and by a series of mechanism-based isocoumarin derivatives, the most potent inhibitor being 4-chloro-7-guanidino-3-(2-phenylethoxy)isocoumarin. Enzyme solutions inactivated by reaction with isocoumarin inhibitors could be completely reactivated after 30 h by treatment with hydroxylamine at neutral pH. Formation of a stable sheep lymph acyl enzyme--in contrast to thrombin and other trypsin-like enzymes--is not followed by alkylation of an active site nucleophile that leads to irreversible enzyme inactivation. The high activity toward substrates with two basic residues suggests that the enzyme may potentially function in processing of precursors of bioactive peptides.     

Interdomain hydrolysis of a truncated Pseudomonas exotoxin by the human immunodeficiency virus-1 protease

The specificity of HIV-1 (human immunodeficiency virus-1) protease has been evaluated relative to its ability to cleave the three-domain Pseudomonas exotoxin (PE66) and related proteins in which the first domain has been deleted or replaced by a segment of CD4. Native PE66 is not hydrolyzed by the HIV-1 protease. However, removal of its first domain produces a molecule which is an excellent substrate for the enzyme. The major site of cleavage in this truncated exotoxin, called LysPE40, occurs in a segment that connects its two major domains, the translocation domain (II), and the ADP-ribosyltransferase (III). This interdomain region contains the sequence ...Asn-Tyr-Pro-Thr... which is similar to that surrounding the scissile Tyr-Pro bond in the gag precursor polyprotein, a natural substrate of the HIV-1 protease. Nevertheless, it is not this sequence that is recognized and cleaved by the enzyme, but one 6 residues away, ...Ala-Leu-Leu-Glu... in which the Leu-Leu peptide bond is hydrolyzed. A second, slower cleavage takes place at the Leu-Ala bond 3 residues in from the NH2 terminus of LysPE40. When domain I of PE66 is replaced by a segment comprising the first two domains of CD4, the resulting chimeric protein is hydrolyzed at the same Leu-Leu bond by HIV-1 protease. Enzyme activities toward synthetic peptides modeled after the sequences defined above in LysPE40 are in complete accord, relative to specificity, kinetics, and pH optimum, with results obtained in the hydrolysis of the parent protein. These findings demonstrate that ideas concerning the specificity of the HIV-1 protease that are based solely upon its processing of natural viral polyproteins can be expanded by evaluation of other multidomain proteins as substrates. Moreover, it would appear that it is not a particular conformation, but sequence and accessibility that play the dominant role in defining sites in a protein substrate that are susceptible to hydrolysis by the enzyme.     

Sbustrate specificity of the elastase and the chymotrypsin-like enzyme of the human granulocyte.

Human granulocyte elastase (EC 3.4.21.11) differs from hog pancreatic elastase in its specificity for synthetic substrates. Although hydrolyzing peptide bonds adjacent to the carboxyl group of alanine, the granulocyte enzyme prefers valine at the cleaved bond, in contrast to the pancreatic enzyme which prefers alanine. Peptide bonds involving the carboxyl group of isoleucine can be hydrolyzed by the granulocyte enzyme but are not hydrolyzed to any significant extent extent by pancreatic elastase. This difference in specificty could explain the lower sensitivity of the granulocyte enzyme to inhibitors containing alanine analogs, such as the peptide chloromethyl ketones and elastatinal. The human granulocyte chymotrypsin-like enzyme differs from pancreatic chymotrypsin by being able to cleave substrates containing leucine in addition to those containing the aromatic amino acids.     

The digestion of the oxidized B chain of insulin by human neutrophile proteases: elastase and chymotrypsin-like protease.

The specificities of human neutrophile elastase and chymotrypsin-like protease towards oxidized insulin B chain were studied. The neutrophile elastase was found to differ from porcine pancreatic elastase in its specificity towards insulin B chain. The neutrophile elastase preferred mostly valine near the cleaved bond in contrast to pancreatic elastase which preferred alanine as well as valine near the cleaved bond. Human neutrophile chymotrypsin-like protease was found to cleave mostly bonds involving leucine and phenylalanine.     

Enzyme:substrate hydrogen bond shortening during the acylation phase of serine protease catalysis

Atomic resolution (相似文献   

Purification and properties of a neutral thiol protease from larval trematode parasite Paragonimus westermani metacercariae

1. A neutral thiol protease was isolated from the extract of larvae of the mammalian trematode parasite, Paragonimus westermani metacercariae, by arginine-Sepharose, Ultrogel AcA-54 and DEAE-toyopearl column chromatography, measuring its activity by the hydrolysis of Boc-Val-Leu-Lys-MCA as a substrate. 2. The molecular weight of the purified enzyme was estimated to be 22,000 as a single polypeptide by SDS-polyacrylamide gel electrophoresis and was estimated to be 20,000 by size exclusion high-performance liquid chromatography. 3. The activity was suppressed by antipain, E-64, leupeptin, chymostatin, N-tosyl-L-lysine chloromethyl ketone, but was not affected by metallo protease inhibitors or serine protease inhibitors. 4. Studies on the substrate specificity showed that the enzyme hydrolyzed Boc-Val-Leu-Lys-MCA, Z-Phe-Arg-MCA, fluorescein isothiocyanate-labeled collagen, azocoll and casein. 5. The enzyme was found to hydrolyze peptide bonds of oxidized insulin B chain preferentially at the carboxy side of hydrophobic and basic amino acids.     

Purification and characterization of a novel metalloendopeptidase from Streptococcus cremoris H61. A metalloendopeptidase that recognizes the size of its substrate

An endopeptidase (LEP-II), which has a unique substrate specificity, was purified to homogeneity by conventional chromatographic techniques from Streptococcus cremoris H61. The enzyme was a metalloendopeptidase since it was inhibited by EDTA and 1,10-phenanthroline; the metal-depleted enzyme could be fully reactivated by micromolar levels of Zn2+ and was not inhibited by specific inhibitors for serine or thiol protease. The molecular mass of the enzyme was estimated to be 80 kDa by Sephacryl S-300 gel filtration and high-performance liquid chromatography with a TSK-G3000SW column. The enzyme consisted of two identical subunits and the N-terminal sequence of LEP-II was determined up to the 19th residue. Although the enzyme had a broad substrate specificity it specifically hydrolyzed the peptide bonds involving the amino groups of hydrophobic amino acid residues. Various small polypeptides, such as alpha s1-CN(f1-23), alpha s1-CN(f91-100), oxidized insulin B chain, glucagon and some biologically active peptides were hydrolyzed. However, a variety of larger polypeptides or proteins, such as alpha s1-CN(f1-54), alpha s1-CN(f61-123), alpha s1-CN(f136-196), alpha s1-casein, beta-casein, and kappa-casein were not hydrolyzed. LEP-II recognized the size of its substrates, which were limited below a molecular mass of about 3.5 kDa.     

Purification and characterization of protease So, a cytoplasmic serine protease in Escherichia coli

A new cytoplasmic endoprotease, named protease So, was purified to homogeneity from Escherichia coli by conventional procedures with casein as the substrate. Its molecular weight was 140,000 when determined by gel filtration on Sephadex G-200 and 77,000 when estimated by polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulfate. Thus, it appears to be composed of two identical subunits. Protease So had an isoelectric point of 6.4 and a K(m) of 1.4 muM for casein. In addition to casein, it hydrolyzed globin, glucagon, and denatured bovine serum albumin to acid-soluble peptides but did not degrade insulin, native bovine serum albumin, or the "auto alpha" fragment of beta-galactosidase. A variety of commonly used peptide substrates for endoproteases were not hydrolyzed by protease So. It had a broad pH optimum of 6.5 to 8.0. This enzyme is a serine protease, since it was inhibited by diisopropyl fluorophosphate and phenylmethylsulfonyl fluoride. Although it was not inhibited by chelating agents, divalent cations (e.g., Mg(2+)) stabilized its activity. Protease So was sensitive to inhibition by N-tosyl-l-phenylalanine chloromethyl ketone but not by N-tosyl-l-lysine chloromethyl ketone. Neither ATP nor 5'-diphosphate-guanosine-3'-diphosphate affected the rate of casein hydrolysis. Protease So was distinct from the other soluble endoproteases in E. coli (including proteases Do, Re, Mi, Fa, La, Ci, and Pi) in its physical and chemical properties and also differed from the membrane-associated proteases, protease IV and V, and from two amino acid esterases, originally named protease I and II. The physiological function of protease So is presently unknown.     

Human pancreatic enzymes: purification and characterization of a nonelastolytic enzyme, protease E. resembling elastase.

?An enzyme with proteolytic activity has been isolated from activated extracts of human pancreatic tissue. The purification procedure included salt fractionation followed by ion-exchange chromatography on SE-TSephadex C-25 and on DEAE-Sephadex A-50. The homogeneity of this enzyme, designated protease te, was demonstrated by disc electrophoresis and by sedimentation equilibrium centrifugation stidues. The homogeneous enzyme shows the ability to hydrolyze many of the conventional synthetic substrates used for the identification of elastase activity; however, it demonstrates no significant elastolytic activity. A comparison of human protease E with porcine elastase reveals a high degree of similarity between the two proteases with respect to inhibition by active-site directed peptide chloromethyl ketones, stability, decreased susceptibility to naturally occurring proteinase inhibitors, and specificity for synthetic substrates as well as several other physical properties. The major difference between human protease E and porcine elastase, other than the lack of elastolytic activity by human protease E, seems to be in the ionic character and the amino acid composition of these two proteins. Porcine elastase is a cationic enzyme, while human protease E appears to be anionic in nature. These dissimilarities concerning elastolytic activity and ionic character appear to be directly related.