Synthetic substrates for human factor VIIa and factor VIIa-tissue factor

A series of 100 tripeptide fluorogenic substrates has been synthesized. These substrates contain Arg in the P1 position, various amino acids in the P2 and P3 positions, and different 6-amino-1-naphthalenesulfonamides (ANSN) as the detecting group (P'). The 38 compounds possessing the highest initial rates of factor VIIa hydrolysis were evaluated for substrate kinetic parameters in the presence and absence of tissue factor (TF) and by factor Xa. Most of these substrates had a higher kcat/KM (keff) value for the factor VIIa-TF complex than for factor Xa. Substitution of different amino acids in the P2 position showed that substrates with bulkier amino acids such as Leu, Pro, and Val have higher values for KM and kcat than those with smaller amino acids (Gly or Ser). The highest second-order rate constants were found for substrates with Val or Pro in the P2 position. A decrease or increase in volume of the P2 substituent (Gly, Ser, or Leu) resulted in a decrease in this constant. Substrates with the highest keff values have Phe in the P3 position. As the hydrophobicity and volume of the amino acid in the P3 position decreased, the keff was reduced. The efficiency of substrates for hydrolysis by factor VIIa was enhanced by an increase of hydrophobicity in the P' structure. TF enhanced the amidolytic activity of the "family" of 38 substrates with ANSN in the P' position on an average of 58-fold.     

Protease substrate specificity mapping using membrane-bound peptides

A method is described for assessing the substrate specificity of proteases by screening for proteolytic activity against large numbers of peptides. All 400 possible peptides derived from the 20 common amino acids were synthesized on small membrane disks in the arrangement FTC-spacer-amino acid P1-amino acid P'1-spacer-membrane, where FTC is a chromophoric group. The disks are incubated simultaneously with the protease, resulting in cleavage of the peptide between the P1 and P'1 amino acids, and the absorbance of the released chromophore is measured as a function of time. As demonstrated for chymotrypsin and papain, plots of the resulting data present a perspective view of the amino acid preferences on both sides of the scissile bond. This technique is fast, requires relatively little enzyme, and can be extended to the systematic screening of longer peptides, including analogs with unnatural amino acids. It has potential use for characterizing the specificity of proteases, assessing the results of site-specific mutagenesis, and searching for optimal substrates and inhibitors.     

Analysis of tissue plasminogen activator specificity using peptidyl fluorogenic substrates

A series of 54 fluorogenic substrates have been synthesized and evaluated for tissue-type plasminogen activator (tPA) hydrolysis in an attempt to create efficient sensitive substrates for tPA and to investigate substrate structure-efficiency correlations. All substrates contain the 6-amino-1-naphthalenesulfonamide (ANSN) leaving group, Arg in the P1 position, various amino acids in the P2 and P3 positions, and various substituents in the sulfonamide moiety of the leaving group (P' position). The majority of substrates have relatively low K(M) values (< 100 microM), reaching as low as 2.6 microM, and reasonably high k(cat) values (up to 3.6 s(-1)). These substrates have higher affinity, higher hydrolysis rates, and higher efficiency for two-chain tPA than for the single-chain form of this enzyme. Analysis of the P3 structure influence on substrate efficiency demonstrates that compounds which contain D-isomers of N-blocked bulky amino acids, such as Phe, Leu, and Val, in this position are more efficient for tPA than substrates with N-unblocked small amino acids (Ser or Pro) in the P3 position. The second-order rate constants and k(cat) values for substrate hydrolysis increase with decreases in the P2 amino acid hydrophobicity in the following manner: Leu < Val and Gly < Ser < Pro. Substrates which contain an ANSN leaving group had a higher affinity for tPA than substrates with p-nitroaniline or 7-amino-4-methylcoumarin leaving groups. Analyses of substrate hydrolysis dependence on the substrate P' structure show that the k(cat) and the second-order rate constants increased with an increase in the size of monoalkyl substituent in the sulfonamide moiety, whereas substrates which contain either glycine methyl ester or a dialkyl group displayed the lowest efficiency for tPA. The substrate Boc-(p-F)Phe-Pro-Arg-ANSNHC2H5 allowed quantitation of tPA at a concentration as low as 1 pM, a concentration significantly lower than the plasma concentration of this protein. Evaluation of the activation of single-chain tPA by factor Xa demonstrates that prothrombinase is approximately 3-fold more efficient in activating sc-tPA than factor Xa alone, increasing the initial rate of activation from 0.0055 nM/s per 1 nM of factor Xa to 0.017 nM/s per 1 nM.     

Random mutagenesis into the conserved Gly154 of subtilisin E: isolation and characterization of the revertant enzymes

We analyzed the role played by the conserved Gly154, a constituent of the P1 substrate-binding pocket of Bacillus subtilis subtilisin E, in the catalytic properties of the protease. Using an Escherichia coli expression system, the termination codon at position 154 in subtilisin E was first introduced to abolish the catalytic activity through truncation of the C-terminus from amino acid residues 154-275. We then attempted to obtain revertants with substitutions of various amino acids at position 154 by the polymerase chain reaction using a mixture of oligonucleotides. In addition to the Gly residue (wild-type), six amino acid substitutions (Ala, Arg, Leu, Phe, Pro and Thr) gave caseinolytic activity. When assayed with synthetic peptide substrates, most of the revertants showed a considerable decrease in specific activity and a P1 specificity similar to that of the wild-type enzyme. An Ala154 mutant purified from the periplasmic space in E. coli, however, resulted in an up to 2.3-fold preference for Val rather than Pro as a P2 substrate relative to the wild-type. Further, a significant 2-10-fold increase in the catalytic efficiency occurred in the Gly127Ala plus Gly154Ala combination variant, relative to the single Gly127Ala variant, without any change in the restricted specificity. The kinetic data and molecular modeling analysis demonstrate the important role of position 154 in the catalytic efficiency as well as in the substrate specificity of subtilisin E.     

Specificity of an extracellular proteinase from Brevibacterium linens ATCC 9174 on bovine alpha s1-casein

The specificity of the extracellular proteinase from Brevibacterium linens ATCC 9174 on bovine alpha s1-casein was studied. Hydrolysis was monitored over time by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (PAGE) and urea-PAGE. The major pH 4.6-soluble peptides were isolated by high-performance liquid chromatography and identified by N-terminal amino acid sequencing and mass spectrometry. The time course of peptide formation indicated that His-8-Gln-9, Ser-161-Gly-162, and either Gln-172-Tyr-173 or Phe-23-Phe-24 were the first, second, and third bonds cleaved, respectively. Other cleavage sites included Asn-19-Leu-20, Phe-32-Gly-33, Tyr-104-Lys-105, Leu-142-Ala-143, Phe-150-Arg-151, Gln-152-Phe-153, Leu-169-Gly-170, and Thr-171-Gln-172. The proteinase had a broad specificity for the amino acid residues at the P1 and P'1 positions but showed a preference for hydrophobic residues at the P2, P3, P4, P'2, P'3, and P'4 positions.     

Elastin degradation by matrix metalloproteinases. Cleavage site specificity and mechanisms of elastolysis

Insoluble elastin was used as a substrate to characterize the peptide bond specificities of human (HME) and mouse macrophage elastase (MME) and to compare these enzymes with other mammalian metalloproteinases and serine elastases. New amino termini detected by protein sequence analysis in insoluble elastin following proteolytic digestion reveal the P'1 residues in the carboxyl-terminal direction from the scissile bond. The relative proportion of each amino acid in this position reflects the proteolytic preference of the elastolytic enzyme. The predominant amino acids detected by protein sequence analysis following cleavage of insoluble elastin with HME, MME, and 92-kDa gelatinase were Leu, Ile, Ala, Gly, and Val. HME and MME were similar in their substrate specificity and showed a stronger preference for Leu/Ile than did the 92-kDa enzyme. Fibroblast collagenase showed no activity toward elastin. The amino acid residues detected in insoluble elastin following hydrolysis with porcine pancreatic elastase and human neutrophil elastase were predominantly Gly and Ala, with lesser amounts of Val, Phe, Ile, and Leu. There were interesting specificity differences between the two enzymes, however. For both the serine and matrix metalloproteinases, catalysis of peptide bond cleavage in insoluble elastin was characterized by temperature effects and water requirements typical of common enzyme-catalyzed reactions, even those involving soluble substrates. In contrast to what has been observed for collagen, the energy requirements for elastolysis were not extraordinary, consistent with cleavage sites in elastin being readily accessible to enzymatic attack.     

Substrate specificity of honeydew melon protease D, a plant serine endopeptidase

The substrate specificity of honeydew melon (Cucumis melo var. inodorus Naud) protease D was studied by the use of synthetic substrates and oligopeptides derived from a protein hydrolyzate. The hydrolysis rates of succinyl-(L-Ala)1-3-p-nitroanilide (Suc-(Ala)1-3-pNA) the hydrolysis rate progressively rose in proportion to the increased chain length. Benzyloxycarbonyl-L-tyrosine p-nitrophenyl ester (Z-Tyr-ONp) and benzoyl-L-tyrosine ethyl ester (Bz-Tyr-OEt) were cleaved by honeydew melon protease D, but benzoyl-L-arginine p-nitroanilide (Bz-Arg-pNA), benzyloxycarbonyl-L-lysine p-nitrophenyl ester (Z-Lys-ONp) and tosyl-L-arginine methyl ester (Tos-Arg-OMe) were not hydrolyzed. Contrary to the results obtained by using synthetic substrates, the carboxyl sides of charged amino acid residues were preferentially cleaved by the enzyme in the oligopeptide substrates. The substrates that had charged or polar amino acids at P2 positions were not cleaved. On the other hand, the non-polar amino acid or proline at P2 were favored for hydrolysis. The information concerning the subsite of protease D was obtained and is useful for synthesis of a good substrate. As it is distinct from molecular mass, the substrate specificity of honeydew melon protease D is most analogous to cucumisin [EC 3.4.21.25] among serine proteases from cucurbitaceous plants.     

Digestion of Luliberine analogues containing D-alanine residue by human gastric juice

The digestion of surphagone and other luliberine analogues containing residues of D-amino acids by human gastric juice was studied. By means of chromatography and mass spectrometry, these peptides were shown to undergo hydrolysis of the bond formed by D-Ala at the P-1 position, and this hydrolysis was shown to be catalyzed by gastricsin rather than pepsin A. Gastricsin was assumed to be a highly specific protease. The results are in a good agreement with the concept of conformational specificity of proteases towards the natural oligopeptides.     

The use of quantitative bacteriologic assessment of bone

A new sub-class of binding protein-dependent transporter with specificity for a broad range of polar amino acids has been identified by sequence comparison, in Rhizobium leguminosarum, Rhodobacter capsulatus, Escherichia coli and Pseudomonas fluorescens. Southern blotting and PCR analysis has shown that transporters from this new sub-class are widely distributed in Gram-negative bacteria, including, in addition to the above, Citrobacter freundii, Erwinia carotovorum and Rhizobium meliloti. ABC transporters of polar amino acids can be divided into two groups: those with narrow solute specificity and the newly identified sub-class with broad solute specificity. The binding and inner membrane proteins from transporters with a broad solute specificity are larger by approximately 30% than those with a narrow solute specificity. Multiple alignment of the inner membrane proteins from all sequenced polar amino acid transporters indicates there is an N-terminal conserved region that may be involved in solute specificity. A conserved arginine or lysine at residue 30 of this region is changed to glutamate in arginine transporters. Residue 53 also has a strong correlation with the charge on the transported solute, with basic amino acid transporters replacing an aliphatic amino acid at this position with a negatively charged amino acid. The general amino acid permease from R. leguminosarum, which will transport aliphatic as well as basic and acidic amino acids, juxtaposes two prolines at residues 52 and 53 of the N-terminal conserved region.     

Purification and characterization of an alkaline elastase from Myxococcus xanthus

An extracellular elastase, termed Myxococcus xanthus alkaline protease 1 (MAP1), has been purified from M. xanthus DK1622 culture supernatants by a combination of ion-exchange and affinity chromatographies. It consists of a single peptide chain of 39 kDa. The elastolytic activity was totally suppressed by 10 mM 1,10-phenanthroline and the enzyme may then be classified as a metalloprotease. Its pH optimum was estimated to be 8.2 with both elastin-orcein and succinyl-Ala3 p-nitroanilide as substrates. Despite its low pI (5.2), MAP1 was adsorbed on elastin at 80%, a result which privileges hydrophobic interactions between MAP1 and elastin rather than salt bridges, as for known basic elastases. About 80% of the original amidasic and elastolytic activities were conserved after a 30-min prior incubation of the enzyme at 40 degrees C; however, 70% of the amidasic activity is measured, instead of 15% for the elastolytic activity, after 30 min at 50 degrees C. Thermal denaturation at this temperature may prevent adsorption of the enzyme on elastin without any important change of the elastase structure. MAP1 readily hydrolyzes the Gly23-Phe24 bond in the oxidized insulin B chain; the peptide bonds Ala14-Leu15, Leu15-Tyr16, Phe24-Phe25, Phe25-Tyr26 are also cleaved, suggesting a primary specificity of the enzyme for hydrophobic or aromatic residues at the first amino acid towards the C-terminus from the cleavage site (P'1 position) [Schechter, I. & Berger, A. (1967) Biochem. Biophys. Res. Commun. 27, 157-162]. This hypothesis is consistent with the fact that Ala2-Phe-Ala and Ala3-Phe-Ala are hydrolyzed even though tri-alanine to hexa-alanine oligomers are not. The evidence of an elastase with the same molecular mass and pI as MAP1 is given during fruiting body development in submerged culture of M. xanthus. The fact that aromatic amino acids have been found to be the most representative of A-signal [Kuspa, A., Plamann, L. & Kaiser, D. (1992) J. Bacteriol. 174, 3319-3326] is consistent with the hypothesis that, regarding its specificity, MAP1 is likely to play a role in development of myxobacteria.     

Cleavage efficiency of the novel aspartic protease yapsin 1 (Yap3p) enhanced for substrates with arginine residues flanking the P1 site: correlation with electronegative active-site pockets predicted by molecular modeling

Yapsin 1, a novel aspartic protease with unique specificity for basic residues, was shown to cleave CCK13-33 at Lys23. Molecular modeling of yapsin 1 identified the active-site cleft to have negative residues close to or within the S6, S3, S2, S1, S1', S2', and S3' pockets and is more electronegative than rhizopuspepsin or endothiapepsin. In particular, the S2' subsite has three negative charges in and close to this pocket that can provide strong electrostatic interactions with a basic residue. The model, therefore, predicts that substrates with a basic residue in the P1 position would be favored with additional basic residues binding to the other electronegative pockets. A deletion of six residues close to the S1 pocket in yapsin 1, relative to rhizopuspepsin and other aspartic proteases of known 3D structure, is likely to affect its specificity. The model was tested using CCK13-33 analogues. We report that yapsin 1 preferentially cleaves a CCK13-33 substrate with a basic residue in the P1 position since the substrates with Ala in P1 were not cleaved. Furthermore, the cleavage efficiency of yapsin 1 was enhanced for CCK13-33 analogues with arginine residues flanking the P1 position. An alanine residue, substituting for the arginine residue in the P6 position in CCK13-33, resulted in a 50% reduction in the cleavage efficiency. Substitution with arginine residues downstream of the cleavage site at the P2', P3', or P6' position increased the cleavage efficiency by 21-, 3- and 7-fold, respectively. Substitution of Lys23 in CCK13-33 with arginine resulted not only in cleavage after the substituted arginine residue, but also forced a cleavage after Met25, suggesting that an arginine residue in the S2' pocket is so favorable that it can affect the primary specificity of yapsin 1. These results are consistent with the predictions from the molecular model of yapsin 1.     

Comparative sequence specificities of human 72- and 92-kDa gelatinases (type IV collagenases) and PUMP (matrilysin)

The sequence specificities of human 72-kDa fibroblast gelatinase (type IV collagenase), human 92-kDa neutrophil gelatinase (type IV collagenase), and putative metalloproteinase (PUMP or matrilysin) have been examined by measuring the rate of hydrolysis of over 50 synthetic oligopeptides covering the P4 through P4' subsites of the substrate. The peptides investigated in this paper were those employed in our previous study which systematically examined the sequence specificity of human fibroblast and neutrophil collagenases [Netzel-Arnett et al. (1991) J. Biol. Chem. 266, 6747]. The initial rate of hydrolysis of the P1-P1' bond of each peptide has been measured under first-order conditions ([S0] < KM), and kcat/KM values have been calculated from the initial rates. The specificities of these five metalloproteinases are similar, but distinct, with the largest differences occurring at subsites P1, P1', and P3'. The specificities of the two gelatinases are the most similar to each other. They tolerate only small amino acids such as Gly and Ala in subsite P1. In contrast, larger residues such as Met, Pro, Gln, and Glu are also accommodated well by PUMP. All five enzymes prefer hydrophobic, aliphatic residues in subsite P1'. PUMP exhibits a stronger preference for Leu in this subsite than is shown by the other enzymes. The P3' subsite specificities of the gelatinases and collagenases are very similar but different from those of PUMP which particularly prefers Met in this position. The specificity data from this study allow the design of optimized substrates and selective inhibitors for these metalloproteinases.     

Hydrophilic peptides derived from the transframe region of Gag-Pol inhibit the HIV-1 protease

The HIV-1 transframe region (TFR) is between the structural and functional domains of the Gag-Pol polyprotein, flanked by the nucleocapsid and the protease domains at its N and C termini, respectively. Transframe octapeptide (TFP) Phe-Leu-Arg-Glu-Asp-Leu-Ala-Phe, the N terminus of TFR, and its analogues are competitive inhibitors of the action of the mature HIV-1 protease. The smallest, most potent analogues are tripeptides: Glu-Asp-Leu and Glu-Asp-Phe with Ki values of approximately 50 and approximately 20 microM, respectively. Substitution of the acidic amino acids in the TFP by neutral amino acids and d or retro-d configurations of Glu-Asp-Leu results in an >40-fold increase in Ki. Protease inhibition by Glu-Asp-Leu is dependent on a protonated form of a group with a pKa of 3.8; unlike other inhibitors of HIV-1 protease which are highly hydrophobic, Glu-Asp-Leu is extremely soluble in water, and its binding affinity decreases with increasing NaCl concentration. However, Glu-Asp-Leu is a poor inhibitor (Ki approximately 7.5 mM) of the mammalian aspartic acid protease pepsin. X-ray crystallographic studies at pH 4.2 show that the interactions of Glu at P2 and Leu at P1 of Glu-Asp-Leu with residues of the active site of HIV-1 protease are similar to those of other product-enzyme complexes. It was not feasible to understand the interaction of intact TFP with HIV-1 protease under conditions of crystal growth due to its hydrolysis giving rise to two products. The sequence-specific, selective inhibition of the HIV-1 protease by the viral TFP suggests a role for TFP in regulating protease function during HIV-1 replication.     

Structural features of autoreactive TCR that determine the degree of degeneracy in peptide recognition

Structural aspects of human TCRs that allow the activation of autoreactive T cells by diverse microbial peptides were examined using two human myelin basic protein (MBP)-specific T cell clones. The TCR sequences of these clones differed only in the N region of TCR-alpha and -beta since the clones had the same Valpha-Jalpha and Vbeta-Jbeta rearrangements. The two clones had a similar fine specificity for the MBP peptide, except for the P5 position of the peptide (lysine). In the crystal structure of the HLA-DR2/MBP peptide complex, P5 lysine is a prominent, solvent-exposed residue in the center of the DR2/MBP peptide surface. Five microbial peptides with conservative or nonconservative changes at the P5 position (lysine to arginine, serine, or proline) activated one of these clones. In contrast, the other clone was activated only by three of these peptides which had a conservative lysine to arginine change at P5. The degree of specificity/degeneracy in recognition of the P5 side chain was the key difference between these TCRs since the Escherichia coli/Haemophilus influenzae peptide stimulated both clones when the P5 position was substituted from serine to arginine. These results demonstrate that the complementarity-determining region 3 loops contribute to the degree of degeneracy in peptide recognition by human MBP-specific TCRs.     

Substrate specificities of pepstatin-insensitive carboxyl proteinases from gram-negative bacteria

Pseudomonas carboxyl proteinase (PCP), isolated from Pseudomonas sp. 101, and Xanthomonas carboxyl proteinase (XCP), isolated from Xanthomonas sp. T-22, are the first and second examples of unique carboxyl proteinases [EC 3.4.23.33] which are insensitive to aspartic proteinase inhibitors, such as pepstatin, diazoacetyl-DL-norleucine methylester, and 1,2-epoxy-3(p-nitrophenoxy)propane. The substrate specificities of PCP and XCP were studied using a series of synthetic chromogenic peptide substrates with the general structure, P5-P4-P3-P2-Phe-Nph-P2'-P3' (P5, P4, P3, P2, P2', P3': a variety of amino acids, Nph is p-nitro-L-phenylalanine, and the Phe-Nph bond is cleaved). PCP and XCP were shown to hydrolyze a synthetic substrate, Lys-Pro-Ala-Leu-Phe-Nph-Arg-Leu, most effectively among 28 substrates. The kinetic parameters of this peptide for PCP were Km = 6.3 microM, Kcat = 51.4 s-1, and kcat/Km = 8.16 microM-1.s-1. The kinetic parameters for XCP were Km = 3.6 microM, kcat = 52.2 s-1, and kcat/Km = 14.5 microM-1.s-1. PCP showed a stricter substrate specificity than XCP. That is, the specificity constant (kcat/Km) of each substrate for PCP was in general < 0.5 microM-1.s-1, but was drastically improved by the replacement of Lys by Leu at the P2 position. On the other hand, XCP showed a less stringent substrate specificity, with most of the peptides exhibiting reasonable kcat/Km values (> 1.0 microM-1.s-1). Thus it was found that the substrate specificities of PCP and XCP differ considerably, in spite of the high similarity in their primary structures. In addition, tyrostatin was found to be a competitive inhibitor for XCP, with a Ki value of 2.1 nM, as well as for PCP (Ki = 2.6 nM).     

Cold-active serine alkaline protease from the psychrotrophic bacterium Shewanella strain ac10: gene cloning and enzyme purification and characterization

The gene encoding serine alkaline protease (SapSh) of the psychrotrophic bacterium Shewanella strain Ac10 was cloned in Escherichia coli. The amino acid sequence deduced from the 2,442-bp nucleotide sequence revealed that the protein was 814 amino acids long and had an estimated molecular weight of 85,113. SapSh exhibited sequence similarities with members of the subtilisin family of proteases, and there was a high level of conservation in the regions around a putative catalytic triad consisting of Asp-30, His-65, and Ser-369. The amino acid sequence contained the following regions which were assigned on the basis of homology to previously described sequences: a signal peptide (26 residues), a propeptide (117 residues), and an extension up to the C terminus (about 250 residues). Another feature of SapSh is the fact that the space between His-65 and Ser-369 is approximately 150 residues longer than the corresponding spaces in other proteases belonging to the subtilisin family. SapSh was purified to homogeneity from the culture supernatant of E. coli recombinant cells by affinity chromatography with a bacitracin-Sepharose column. The recombinant SapSh (rSapSh) was found to have a molecular weight of about 44,000 and to be highly active in the alkaline region (optimum pH, around 9.0) when azocasein and synthetic peptides were used as substrates. rSapSh was characterized by its high levels of activity at low temperatures; it was five times more active than subtilisin Carlsberg at temperatures ranging from 5 to 15 degreesC. The activation energy for hydrolysis of azocasein by rSapSh was much lower than the activation energy for hydrolysis of azocasein by the subtilisin. However, rSapSh was far less stable than the subtilisin.     

Programming the Rous sarcoma virus protease to cleave new substrate sequences

The Rous sarcoma virus protease displays a high degree of specificity and catalyzes the cleavage of only a limited number of amino acid sequences. This specificity is governed by interactions between side chains of eight substrate amino acids and eight corresponding subsite pockets within the homodimeric enzyme. We have examined these complex interactions in order to learn how to introduce changes into the retroviral protease (PR) that direct it to cleave substrates. Mutant enzymes with altered substrate specificity and wild-type or greater catalytic rates have been constructed previously by substituting single key amino acids in each of the eight enzyme subsites with those residues found in structurally related positions of human immunodeficiency virus (HIV)-1 PR. These individual amino acid substitutions have now been combined into one enzyme, resulting in a highly active mutant Rous sarcoma virus (RSV) protease that displays many characteristics associated with the HIV-1 enzyme. The hybrid protease is capable of catalyzing the cleavage of a set of HIV-1 viral polyprotein substrates that are not recognized by the wild-type RSV enzyme. Additionally, the modified PR is inhibited completely by the HIV-1 PR-specific inhibitor KNI-272 at concentrations where wild-type RSV PR is unaffected. These results indicate that the major determinants that dictate RSV and HIV-1 PR substrate specificity have been identified. Since the viral protease is a homodimer, the rational design of enzymes with altered specificity also requires a thorough understanding of the importance of enzyme symmetry in substrate selection. We demonstrate here that the enzyme homodimer acts symmetrically in substrate selection with each enzyme subunit being capable of recognizing both halves of a peptide substrate equally.     

P1 specificity of aqualysin I (a subtilisin-type serine protease) from Thermus aquaticus YT-1, using P1-substituted derivatives of Streptomyces subtilisin inhibitor

Aqualysin I is an alkaline serine protease isolated from Thermus aquaticus YT-1, an extreme thermophile. We have measured the P1-specificity of aqualysin I, using wild-type and five P1-substituted derivatives of Streptomyces subtilisin inhibitor (SSI). SSIs efficiently inhibited the activity of aqualysin I, with low substrate specificity. Charge and hydrophobicity of side chain of the P1 amino acid residue showed no significant effect to the P1-specificity of this enzyme.     

Membrane type-1 matrix metalloprotease and stromelysin-3 cleave more efficiently synthetic substrates containing unusual amino acids in their P1' positions

The influence of the substrate P1' position on the specificity of two zinc matrix metalloproteases, membrane type-1 matrix metalloprotease (MT1-MMP) and stromelysin-3 (ST3), was evaluated by synthesizing a series of fluorogenic substrates of general formula dansyl-Pro-Leu-Ala-Xaa-Trp-Ala-Arg-NH2, where Xaa in the P1' position represents unusual amino acids containing either long arylalkyl or alkyl side chains. Our data demonstrate that both MT1-MMP and ST3 cleave substrates containing in their P1' position unusual amino acids with extremely long side chains more efficiently than the corresponding substrates with natural phenylalanine or leucine amino acids. In this series of substrates, the replacement of leucine by S-para-methoxybenzyl cysteine increased the kcat/Km ratio by a factor of 37 for MT1-MMP and 9 for ST3. The substrate with a S-para-methoxybenzyl cysteine residue in the P1' position displayed a kcat/Km value of 1.59 10(6) M-1 s-1 and 1.67 10(4) M-1 s-1, when assayed with MT1-MMP and ST3, respectively. This substrate is thus one of the most rapidly hydrolyzed substrates so far reported for matrixins, and is the first synthetic peptide efficiently cleaved by ST3. These unexpected results for these two matrixins suggest that extracellular proteins may be cleaved by matrixins at sites containing amino acids with unusual long side chains, like those generated in vivo by some post-translational modifications.