Atrial dipeptidyl carboxyhydrolase is a zinc-metallo proteinase which possesses tripeptidyl carboxyhydrolase activity

Atrial dipeptidyl carboxyhydrolase readily converts one atrial natriuretic peptide, atriopeptin II (Ser103-Arg125 peptide), to another, atriopeptin I (Ser103-Ser123 peptide), by selective removal of the C-terminal dipeptide, Phe-Arg. The atrial peptides possess natriuretic, diuretic, smooth muscle relaxant, and cardiodynamic properties and their existence has shown the mammalian heart to be an endocrine organ. After inactivating the bovine atrial enzyme with EDTA, activity is restored by the addition of Co+2, Zn+2 and Mn+2 but not by Cu+2, Mg+2, Ca+2, or Cd+2. The enzyme is thus likely to be a zinc-metallo proteinase. In addition to its dipeptidyl activity, the enzyme also displays tripeptidyl carboxyhydrolase activity with atriopeptin III (Ser103-Try126 peptide) as substrate. The hydrolytic products resulting from tripeptidyl cleavage are atriopeptin I and Phe-Arg-Tyr. However, with [mercaptopropionyl105,(D)Ala107]-atriopeptin III-NH2 peptide (a potent agonist of atriopeptin III) as substrate, the enzyme acts exclusively as a tripeptidyl carboxyhydrolase. To examine the basis for this shift in cleavage point, pentapeptides based on the C-terminal sequence of atriopeptin III were prepared; a C-terminal Tyr or Tyr-NH2 residue is not sufficient to cause the change in cleavage point. The amidated pentapeptide is not a substrate but is a competitive inhibitor of hydrolysis of the corresponding free-acid peptide.     

Purification and characterization of two serine carboxypeptidases from Aspergillus niger and their use in C-terminal sequencing of proteins and peptide synthesis.

A procedure was developed to prepare in large amounts two carboxypeptidases, CPD-I and CPD-II, from Aspergillus niger. They were each shown to be serine proteases and single-chain monomers with molecular masses of ca. 81 kDa and containing 22% carbohydrates. Amino acid analysis, carbohydrate determination, and N-terminal sequencing (20 to 25 residues) were performed on each enzyme. CPD-I showed sequence homologies with malt carboxypeptidase II, while the N terminus of CPD-II was different from that of any known serine carboxypeptidase. Like carboxypeptidase Y from Saccharomyces cerevisiae and carboxypeptidase III from malt, CPD-II contained a free sulfhydryl group that could play a role in catalysis. Both A. niger enzymes had pH optima of about 4 and were unstable above pH 7. Their specificities for substrate positions P1 and P'1 were characterized by use of, as substrates, a series of N-blocked amino acid esters and dipeptides. Both enzymes were specific for Arg, Lys, and Phe in P1. CPD-I preferred hydrophobic residues in P'1, while CPD-II was highly specific for Arg and Lys in this position. Each displayed an original specificity when P1 and P'1 were considered together. The specificities were also studied by analyzing the time course of the release of amino acids from eight different peptides of various lengths. CPD-I and CPD-II appeared to be quite suitable for C-terminal sequence studies as well as for the synthesis of peptide bonds. The latter was studied with two peptide esters as aminolysis substrates and a series of amino acid amides as nucleophiles.(ABSTRACT TRUNCATED AT 250 WORDS)     

Mapping of human plasma kallikrein active site by design of peptides based on modifications of a Kazal-type inhibitor reactive site

Human plasma kallikrein (huPK) is a proteinase that participates in several biological processes. Although various inhibitors control its activity, members of the Kazal family have not been identified as huPK inhibitors. In order to map the enzyme active site, we synthesized peptides based on the reactive site (PRILSPV) of a natural Kazal-type inhibitor found in Cayman plasma, which is not an huPK inhibitor. As expected, the leader peptide (Abz-SAPRILSPVQ-EDDnp) was not cleaved by huPK. Modifications to the leader peptide at P'1, P'3 and P'4 positions were made according to the sequence of a phage display-generated recombinant Kazal inhibitor (PYTLKWV) that presented huPK-binding ability. Novel peptides were identified as substrates for huPK and related enzymes. Both porcine pancreatic and human plasma kallikreins cleaved peptides at Arg or Lys bonds, whereas human pancreatic kallikrein cleaved bonds involving Arg or a pair of hydrophobic amino acid residues. Peptide hydrolysis by pancreatic kallikrein was not significantly altered by amino acid replacements. The peptide Abz-SAPRILSWVQ-EDDnp was the best substrate and a competitive inhibitor for huPK, indicating that Trp residue at the P'4 position is important for enzyme action.     

Purification and characterization of two serine carboxypeptidases from Aspergillus niger and their use in C-terminal sequencing of proteins and peptide synthesis.

A procedure was developed to prepare in large amounts two carboxypeptidases, CPD-I and CPD-II, from Aspergillus niger. They were each shown to be serine proteases and single-chain monomers with molecular masses of ca. 81 kDa and containing 22% carbohydrates. Amino acid analysis, carbohydrate determination, and N-terminal sequencing (20 to 25 residues) were performed on each enzyme. CPD-I showed sequence homologies with malt carboxypeptidase II, while the N terminus of CPD-II was different from that of any known serine carboxypeptidase. Like carboxypeptidase Y from Saccharomyces cerevisiae and carboxypeptidase III from malt, CPD-II contained a free sulfhydryl group that could play a role in catalysis. Both A. niger enzymes had pH optima of about 4 and were unstable above pH 7. Their specificities for substrate positions P1 and P'1 were characterized by use of, as substrates, a series of N-blocked amino acid esters and dipeptides. Both enzymes were specific for Arg, Lys, and Phe in P1. CPD-I preferred hydrophobic residues in P'1, while CPD-II was highly specific for Arg and Lys in this position. Each displayed an original specificity when P1 and P'1 were considered together. The specificities were also studied by analyzing the time course of the release of amino acids from eight different peptides of various lengths. CPD-I and CPD-II appeared to be quite suitable for C-terminal sequence studies as well as for the synthesis of peptide bonds. The latter was studied with two peptide esters as aminolysis substrates and a series of amino acid amides as nucleophiles.(ABSTRACT TRUNCATED AT 250 WORDS)     

Degradation of a signal peptide by protease IV and oligopeptidase A.

The degradation of the prolipoprotein signal peptide in vitro by membranes, cytoplasmic fraction, and two purified major signal peptide peptidases from Escherichia coli was followed by reverse-phase liquid chromatography (RPLC). The cytoplasmic fraction hydrolyzed the signal peptide completely into amino acids. In contrast, many peptide fragments accumulated as final products during the cleavage by a membrane fraction. Most of the peptides were similar to the peptides formed during the cleavage of the signal peptide by the purified membrane-bound signal peptide peptidase, protease IV. Peptide fragments generated during the cleavage of the signal peptide by protease IV and a cytoplasmic enzyme, oligopeptidase A, were identified from their amino acid compositions, their retention times during RPLC, and knowledge of the amino acid sequence of the signal peptide. Both enzymes were endopeptidases, as neither dipeptides nor free amino acids were formed during the cleavage reactions. Protease IV cleaved the signal peptide predominantly in the hydrophobic segment (residues 7 to 14). Protease IV required substrates with hydrophobic amino acids at the primary and the adjacent substrate-binding sites, with a minimum of three amino acids on either side of the scissile bond. Oligopeptidase A cleaved peptides (minimally five residues) that had either alanine or glycine at the P'1 (primary binding site) or at the P1 (preceding P'1) site of the substrate. These results support the hypothesis that protease IV is the major signal peptide peptidase in membranes that initiates the degradation of the signal peptide by making endoproteolytic cuts; oligopeptidase A and other cytoplasmic enzymes further degrade the partially degraded portions of the signal peptide that may be diffused or transported back into the cytoplasm from the membranes.     

The specificity of carboxypeptidase Y may be altered by changing the hydrophobicity of the S'1 binding pocket.

The S'1 binding pocket of carboxypeptidase Y is hydrophobic, spacious, and open to solvent, and the enzyme exhibits a preference for hydrophobic P'1 amino acid residues. Leu272 and Ser297, situated at the rim of the pocket, and Leu267, slightly further away, have been substituted by site-directed mutagenesis. The mutant enzymes have been characterized kinetically with respect to their P'1 substrate preferences using the substrate series FA-Ala-Xaa-OH (Xaa = Leu, Glu, Lys, or Arg) and FA-Phe-Xaa-OH (Xaa = Ala, Val, or Leu). The results reveal that hydrophobic P'1 residues bind in the vicinity of residue 272 while positively charged P'1 residues interact with Ser297. Introduction of Asp or Glu at position 267 greatly reduced the activity toward hydrophobic P'1 residues (Leu) and increased the activity two- to three-fold for the hydrolysis of substrates with Lys or Arg in P'1. Negatively charged substituents at position 272 reduced the activity toward hydrophobic P'1 residues even more, but without increasing the activity toward positively charged P'1 residues. The mutant enzyme L267D + L272D was found to have a preference for substrates with C-terminal basic amino acid residues. The opposite situation, where the positively charged Lys or Arg were introduced at one of the positions 267, 272, or 297, did not increase the rather low activity toward substrates with Glu in the P'1 position but greatly reduced the activity toward substrates with C-terminal Lys or Arg due to electrostatic repulsion. The characterized mutant enzymes exhibit various specificities, which may be useful in C-terminal amino acid sequence determinations.     

Sequence specificities of human fibroblast and neutrophil collagenases.

The sequence specificities of human fibroblast and neutrophil collagenases have been investigated by measuring the rate of hydrolysis of 60 synthetic oligopeptides covering the P4 through P'5 subsites of the substrate. The choice of peptides was patterned after both known cleavage sites in noncollagenous proteins and potential cleavage sites (those containing Gly-Ile-Ala, Gly-Leu-Ala, or Gly-Ile-Leu sequences) found in types I, II, III, and IV collagens. The initial rate of hydrolysis of the P1-P'1 bond of each peptide has been measured under first-order conditions ([SO] much less than KM), and kcat/KM values have been calculated from the initial rates. The amino acids in subsites P4 through P'4 all influence the hydrolysis rates for both collagenases. However, the effects of substitutions at each site are distinctive and are consistent with the view that human fibroblast and neutrophil collagenases are homologous but nonidentical enzymes. For peptides with unblocked NH2 and COOH termini, occupancy of subsites P3 through P'3 is necessary for rapid hydrolysis. Compared with the alpha 1(I) cleavage sequence, none of the substitutions investigated at subsites P3, P2, and P'4 produces markedly improved substrates. In contrast, many substitutions at subsites P1, P'1, and P'2 improve specificity. The preferences of both collagenases for alanine in subsite P1 and tryptophan or phenylalanine in subsite P'2, is noteworthy. Human neutrophil collagenase accommodates aromatic residues in subsite P'1 much better than human fibroblast collagenase. The subsite preferences observed for human fibroblast collagenase in these studies agree well with the residues found at cleavage sites in noncollagenous substrates. However, the sequence specificities of these collagenases cannot explain the failure of these enzymes to hydrolyze many potentially cleavable but apparently protected sites in intact collagens. This represents additional support for the notion that the local structure of collagen is important in determining the location of collagenase cleavage sites.     

Mechanism of proline-specific proteinases: (I) Substrate specificity of dipeptidyl peptidase IV from pig kidney and proline-specific endopeptidase from Flavobacterium meningosepticum

The substrate specificity of dipeptidyl peptidase IV (dipeptidyl peptide hydrolase, EC 3.4.14.5) from pig kidney and proline-specific endopeptidase from Flavobacterium meningosepticum, was investigated with a series of N-terminal unprotected (dipeptidyl peptidases IV) and succinylated dipeptidyl-p-nitroanilides (proline-specific endopeptidase). Both enzymes are specific for the S configuration of the amino-acid residue in P1 and P2 position if the penultimate residue is proline. In the case of alanine substrates (Ala in P1, dipeptidyl peptidase IV hydrolyzes such compounds where the configuration of the P2 residue is R. The penultimate residue with dipeptidyl peptidase IV can be, beside proline and alanine, dehydroproline, hydroxyproline and pipecolic acid. Proline substrates (Pro in P1) with an R configuration in P2 are inhibitors of the hydrolysis of proline substrates with an S,S configuration in an uncompetitive (dipeptidyl peptide IV) or mixed inhibition type (proline-specific endopeptidase). Derivatives of Gly-Pro-pNA where the N-terminal amino group is methylated are hydrolyzed by dipeptidyl peptidase IV.     

New serine carboxypeptidase in mung bean seedling cotyledons

Two serine carboxypeptidases (EC 3.4.16.5) were purified from mung bean seedling cotyledons. Sequences of tryptic peptides derived from the 42.5 kD enzyme corresponded to the derived amino acid sequence of a sequenced cDNA (GenBank U49382 and U49741). This enzyme exhibited the substrate specificity pattern previously published for mung bean carboxypeptidase I. In comparison, the sequence and substrate specificity data obtained for the 43 kD enzyme were similar but not identical. Both enzymes showed preference for peptide substrates with a large hydrophobic residue at the C-terminus. With regard to the penultimate residue of peptide substrates, the mung bean carboxypeptidase I preferred small aliphatic amino acid residues, while the 43 kD enzyme preferred large hydrophobic ones.     

Altering catalytic properties of 3-chlorocatechol-oxidizing extradiol dioxygenase from Sphingomonas xenophaga BN6 by random mutagenesis

The 2,3-dihydroxybiphenyl 1,2-dioxygenase from Sphingomonas xenophaga strain BN6 (BphC1) oxidizes 3-chlorocatechol by a rather unique distal ring cleavage mechanism. In an effort to improve the efficiency of this reaction, bphC1 was randomly mutated by error-prone PCR. Mutants which showed increased activities for 3-chlorocatechol were obtained, and the mutant forms of the enzyme were shown to contain two or three amino acid substitutions. Variant enzymes containing single substitutions were constructed, and the amino acid substitutions responsible for altered enzyme properties were identified. One variant enzyme, which contained an exchanged amino acid in the C-terminal part, revealed a higher level of stability during conversion of 3-chlorocatechol than the wild-type enzyme. Two other variant enzymes contained amino acid substitutions in a region of the enzyme that is considered to be involved in substrate binding. These two variant enzymes exhibited a significantly altered substrate specificity and an about fivefold-higher reaction rate for 3-chlorocatechol conversion than the wild-type enzyme. Furthermore, these variant enzymes showed the novel capability to oxidize 3-methylcatechol and 2,3-dihydroxybiphenyl by a distal cleavage mechanism.     

Rapid optimization of enzyme substrates using defined substrate mixtures.

A strategy is described for the rapid optimization of kcat/Km for protease substrates. Selected positions of a given peptide substrate sequence are varied through synthesis with mixtures of amino acids. Incubation of the resulting peptide mixture with the enzyme of interest and analysis by high pressure liquid chromatography provides a direct measure of analogs with enhanced kcat/Km. High performance liquid chromatography/continuous flow fast atom bombardment mass spectrometry is used to assign structure to each peak in the chromatogram. As an example of the utility and efficiency of "substrate mapping" we describe optimization of the collagenase substrate Dnp-Pro-Leu-Gly-Leu-Trp-Ala-D-Arg-NH2 (where Dnp is dinitrophenyl) at the P'1 and P'2 positions. Six different mixtures were prepared for evaluation, representing the synthesis of 128 different synthetic substrates. "Substrate mapping" has led to Dnp-Pro-Leu-Gly-Cys(Me)-His-Ala-D-Arg-NH2, a substrate that possesses a 10-fold better kcat/Km than Dnp-Pro-Leu-Gly-Leu-Trp-Ala-D-Arg-NH2.     

Enzyme-substrate interactions in the hydrolysis of peptide substrates by thermitase, subtilisin BPN', and proteinase K

Peptide substrates of the general structure acetyl-Alan (n = 2-5), acetyl-Pro-Ala-Pro-Phe-Alan-NH2 (n = 0-3), and acetyl-Pro-Ala-Pro-Phe-AA-NH2 (AA = various amino acids) were synthesized and used to investigate the enzyme-substrate interactions of the microbial serine proteases thermitase, subtilisin BPN', and proteinase K on the C-terminal side of the scissile bond. The elongation of the substrate peptide chain up to the second amino acid on the C-terminal side (P'2) enhances the hydrolysis rate of thermitase and subtilisin BPN', whereas for proteinase K an additional interaction with the third amino acid (P'3) is possible. The enzyme subsite S'1 specificity of the proteases investigated is very similar. With respect to kcat/Km values small amino acid residues such as Ala and Gly are favored in this position. Bulky residues such as Phe and Leu were hydrolyzed to a lower extent. Proline in P'1 abolishes the hydrolysis of the substrates. Enzyme-substrate interactions on the C-terminal side of the scissile bond appear to affect kcat more than Km for all three enzymes.     

Favourable side-chain orientation of cleavage site dibasic residues of prohormone in proteolytic processing by prohormone convertase 1/3.

Previous studies using selectively modified pro-ocytocin/neurophysin substrate analogues and the purified metalloprotease, pro-ocytocin/neurophysin convertase (magnolysin; EC 3.4 24.62), have shown that dibasic cleavage site processing is associated with a prohormone sequence organized in a beta-turn structure. We have used various peptide analogues of the pro-ocytocin-neurophysin processing domain, and recombinant prohormone convertase 1/3, to test the validity of this property towards this member of the family of prohormone convertases (PCs). The enzymatic cleavage analysis and kinetics showed that: (a) with methyl amide (N-Met) modification, a secondary structure beta-turn breaker, the enzyme substrate interaction was abolished; (b) cleavage was favoured when the dibasic substrate side-chains were oriented in opposite directions; (c) the amino acid present at the P'1 position is important in the enzyme-substrate interaction; (d) the flexibility of the peptide substrate is necessary for the interaction; (e) Addition of dimethylsulfoxide to the cleavage assay favoured the cleavage of the pro-ocytocin/neurophysin large substrate over that of the smaller one pGlu-Arg-Thr-Lys-Arg-methyl coumarin amide. These data allowed us to conclude that proteolytic processing of pro-ocytocin-related peptide substrates by PC1/3 as well as by the metalloenzyme, magnolysin, involves selective recognition of precise cleavage site local secondary structure by the processing enzyme. It is hypothesized that this may represent a general property of peptide precursor proteolytic processing systems.     

Substrate Specificity and Possible Heterologous Targets of Phytaspase,a Plant Cell Death Protease

Plants lack aspartate-specific cell death proteases homologous to animal caspases. Instead, a subtilisin-like serine-dependent plant protease named phytaspase shown to be involved in the accomplishment of programmed death of plant cells is able to hydrolyze a number of peptide-based caspase substrates. Here, we determined the substrate specificity of rice (Oryza sativa) phytaspase by using the positional scanning substrate combinatorial library approach. Phytaspase was shown to display an absolute specificity of hydrolysis after an aspartic acid residue. The preceding amino acid residues, however, significantly influence the efficiency of hydrolysis. Efficient phytaspase substrates demonstrated a remarkable preference for an aromatic amino acid residue in the P3 position. The deduced optimum phytaspase recognition motif has the sequence IWLD and is strikingly hydrophobic. The established pattern was confirmed through synthesis and kinetic analysis of cleavage of a set of optimized peptide substrates. An amino acid motif similar to the phytaspase cleavage site is shared by the human gastrointestinal peptide hormones gastrin and cholecystokinin. In agreement with the established enzyme specificity, phytaspase was shown to hydrolyze gastrin-1 and cholecystokinin at the predicted sites in vitro, thus destroying the active moieties of the hormones.     

Kinetics and sequence specificity of processing of prepilin by PilD, the type IV leader peptidase of Pseudomonas aeruginosa.

PilD, originally isolated as an essential component for the biogenesis of the type IV pili of Pseudomonas aeruginosa, is a unique endopeptidase responsible for processing the precursors of the P. aeruginosa pilin subunits. It is also required for the cleavage of the leader peptides from the Pdd proteins, which are essential components of an extracellular secretion pathway specific for the export of a number of P. aeruginosa hydrolytic enzymes and toxins. Substrates for PilD are initially synthesized with short, i.e., 6- to 8-amino-acid-long, leader peptides with a net basic charge and share a high degree of amino acid homology through the first 16 to 30 residues at the amino terminus. In addition, they all have a phenylalanine residue at the +1 site relative to the cleavage site, which is N methylated prior to assembly into the oligomeric structures. In this study, the kinetics of leader peptide cleavage from the precursor of the P. aeruginosa pilin subunit by PilD was determined in vitro. The rates of cleavage were compared for purified enzyme and substrate as well as for enzyme and substrate contained within total membranes extracted from P. aeruginosa strains overexpressing the cloned pilD or pilA genes. Optimal conditions were obtained only when both PilD and substrate were contained within total membranes. PilD catalysis of P. aeruginosa prepilin followed normal Michaelis-Menten kinetics, with a measured apparent Km of approximately 650 microM, and a kcat of 180 min-1. The kinetics of PilD processing of another type IV pilin precursor, that from Neisseria gonorrhoeae with a 7-amino-acid-long leader peptide, were essentially the same as that measured for wild-type P. aeruginosa prepilin. Quite different results were obtained for a number of prepilin substrates containing substitutions at the conserved phenylalanine at the +1 position relative to the cleavage site, which were previously shown to be well tolerated in vivo. Substitutions of methionine, serine, and cysteine for phenylalanine show that Km values remain close to that measured for wild-type substrate, while kcat and kcat/Km values were significantly decreased. This indicates that while the affinity of enzyme for substrate is relatively unaffected by the substitutions, the maximum rate of catalysis favors a phenylalanine at this position. Interesting, PilD cleavage of one mutated pillin (asparagine) resulted in a lower Km value of 52.5 microM, which indicates a higher affinity for the enzyme, as well as a lower kcat value of 6.1 min m(-1). This suggests that it may be feasible to design peptide inhibitors of PilD.     

Partial sequence data for the L-(+)-lactate dehydrogenase from Streptococcus cremoris US3 including the amino acid sequences around the single cysteine residue and at the N-terminus

The following amino acid sequence information has been determined for the fructose 1,6-bisphosphate-dependent lactate dehydrogenase from Streptococcus cremoris US3: the C-terminal amino acid, the N-terminal sequence of the first 20 amino acids and the sequence of a 53-residue tryptic peptide containing the only cysteine residue in the protein. The enzyme was cleaved by alkali at the cysteine residue following reaction first with 5,5'-dithiobis(2-nitrobenzoic acid) and then with K14CN. This treatment yielded two cleavage products as well as some higher polymers and some uncleaved enzyme. The radioactive cleavage product was purified and its size indicated that the cysteine residue is 80 residues from the C-terminus. Comparisons of the sequences determined for the S. cremoris enzyme with those already known for dogfish lactate dehydrogenase indicate that the two enzymes are only distantly related since the sequence homology between them is limited and of borderline statistical significance.     

Substrate requirements of human rhinovirus 3C protease for peptide cleavage in vitro

A series of synthetic peptides representing authentic proteolytic cleavage sites of human rhinovirus type 14 were assayed as substrates for purified 3C protease. Competition cleavage assays were employed to determine the relative specificity constants (Kcat/Km) for substrates with sequences related to the viral 2C-3A cleavage site. Variable length peptides representing the 2C-3A cleavage site were cleaved with comparable efficiency. These studies defined a minimum substrate of 6 amino acids (TLFQ/GP), although retention of the residue at position P5 (ETLFQ/GP) resulted in a better substrate by an order of magnitude. Amino acid substitutions at position P5, P4, P1', or P2' indicated that the identity of the residue at position P5 was not critical, whereas substitutions at position P4, P1' or P2' resulted in substrates with Kcat/Km values varying over 2 orders of magnitude. In contrast to the 2C-3A cleavage site, small peptide derivatives representative of the 3A-3B cleavage site were relatively poor substrates, which suggested that residues flanking the minimum core sequence may influence susceptibility to cleavage. The 3C protease of rhinovirus type 14 was also capable of cleaving peptides representing comparable cleavage sites predicted for coxsackie B virus and poliovirus.     

Mutant subtilisin E with enhanced protease activity obtained by site-directed mutagenesis

The specific activity of subtilisin E, an alkaline serine protease of Bacillus subtilis, was substantially increased by optimizing the amino acid residue at position 31 (Ile in the wild-type enzyme) in the vicinity of the catalytic triad of the enzyme. Eight uncharged amino acids (Cys, Ser, Thr, Gly, Ala, Val, Leu, and Phe) were introduced at this site, which is next to catalytic Asp32, using site-directed mutagenesis. Mutant enzymes were expressed in Escherichia coli and were prepared from the periplasmic space. Only the Val and Leu substitutions gave active enzyme, and the Leu31 mutant was found to have a greatly increased activity compared to the wild-type enzyme. The other six mutant enzymes showed a marked decrease in activity. This result indicates that a branched-chain amino acid at position 31 is essential for the expression of subtilisin activity and that the level of the activity depends on side chain structure. The purified Leu31 mutant enzyme was analyzed with respect to substrate specificity, heat stability, and optimal temperature. It was found that the Leu31 replacement caused a prominent 2-6-fold increase in catalytic efficiency (kcat/Km) due to a larger kcat for peptide substrates.     

Characterization of the specificity of arginine-specific gingipains from Porphyromonas gingivalis reveals active site differences between different forms of the enzymes

Porphyromonas gingivalis is a pathogen associated with periodontal disease, and arginine-specific proteases (gingipains-R) from the bacterium are important virulence factors. The specificity of two forms of gingipain-R, HRgpA and RgpB, for substrate positions C-terminal to the cleavage site was analyzed, and notable differences were observed between the enzymes. Molecular modeling of the HRgpA catalytic domain, based on the structure of RgpB, revealed that there are four amino acid substitutions around the active site of HRgpA relative to RgpB that may explain their different specificity. Previously, differences in the ability of these two gingipain-R forms to cleave a number of proteins were attributed to additional adhesins on HRgpA mediating increased interaction with the substrates. Here, purified RgpA(cat), the catalytic domain of HRgpA, which like RgpB also lacks adhesin subunits, was used to show that the differences between HRgpA and RgpB are probably due to the amino acid substitutions at the active site. The kinetics of cleavage of fibrinogen, a typical protein substrate for the gingipain-R enzymes, which is bound by HRgpA but not RgpA(cat) or RgpB, were evaluated, and it was shown that there was no difference in the cleavage of the fibrinogen Aalpha-chain between the different enzyme forms. HRgpA degraded the fibrinogen Bbeta-chain more efficiently, generating distinct cleavage products. This indicates that while the adhesin domain(s) play(s) a minor role in the cleavage of protein substrates, the major effect is still provided by the amino acid substitutions at the active site of rgpA gene products versus those of the rgpB gene.     

Continuous fluorogenic substrates for atrial dipeptidyl carboxyhydrolase. Importance of Ser in the P1 position

Several N-acyltetrapeptides of the general structure 2-aminobenzoyl-Gly-X-Phe(4-nitro)-Arg were synthesized and tested as substrates for atrial dipeptidyl carboxyhydrolase, an enzyme associated with atrial granules that converts one active atrial natriuretic peptide, atriopeptin II, to another, atriopeptin I. Hydrolysis of the X-Phe(4-nitro) bond generates the 2-aminobenzoyl fluorophore and the increasing fluorescence can be monitored in a continuous assay. Based on the ratio of Vmax/Km as an indication of substrate specificity, peptides containing X = Ser greater than Ala approximately equal to Lys- greater than Asn much greater than Thr approximately equal to Asp. With the exception of the Asn substrate, the Km determined for all the substrates was about the same. Thus, the effect of the P1 residue substitution shows up almost exclusively in Vmax.