Mechanism of inhibition of the retroviral protease by a Rous sarcoma virus peptide substrate representing the cleavage site between the gag p2 and p10 proteins.

The activity of the avian myeloblastosis virus (AMV) or the human immunodeficiency virus type 1 (HIV-1) protease on peptide substrates which represent cleavage sites found in the gag and gag-pol polyproteins of Rous sarcoma virus (RSV) and HIV-1 has been analyzed. Each protease efficiently processed cleavage site substrates found in their cognate polyprotein precursors. Additionally, in some instances heterologous activity was detected. The catalytic efficiency of the RSV protease on cognate substrates varied by as much as 30-fold. The least efficiently processed substrate, p2-p10, represents the cleavage site between the RSV p2 and p10 proteins. This peptide was inhibitory to the AMV as well as the HIV-1 and HIV-2 protease cleavage of other substrate peptides with Ki values in the 5-20 microM range. Molecular modeling of the RSV protease with the p2-p10 peptide docked in the substrate binding pocket and analysis of a series of single-amino acid-substituted p2-p10 peptide analogues suggested that this peptide is inhibitory because of the potential of a serine residue in the P1' position to interact with one of the catalytic aspartic acid residues. To open the binding pocket and allow rotational freedom for the serine in P1', there is a further requirement for either a glycine or a polar residue in P2' and/or a large amino acid residue in P3'. The amino acid residues in P1-P4 provide interactions for tight binding of the peptide in the substrate binding pocket.     

Selective cleavage of peptide bonds by cathepsins L and B from rat liver

The selective cleavage of peptide bonds by cathepsin L from rat liver was examined with a hexapeptide, luteinizing hormone releasing hormone, neurotensin and oxidized insulin A chain as model substrates. The specificity of cathepsin L was compared with that of cathepsin B. Cathepsin L cleaved peptide bonds that have a hydrophobic amino acid, such as Phe, Leu, Val, and Trp or Tyr, in position P2. A polar amino acid, such as Tyr, Ser, Gly, Glu, Asp, Gln, or Asn, in position P1. enhanced the susceptibility of the peptide bond to cathepsin L, though the importance of the amino acid residue in position P1' was not as great as that of the amino acid in position P2 for the action of cathepsin L. These results suggest that, in contrast to cathepsin B, cathepsin L shows very clear specificity.     

Proline-specific endopeptidases from microbial sources: isolation of an enzyme from a Xanthomonas sp.

An extensive screening among microorganisms for the presence of post-proline-specific endopeptidase activity was performed. This activity was found among ordinary bacteria from soil samples but not among fungi and actinomycetes. This result is in contrast to the previous notion that this activity is confined to the genus Flavobacterium. A proline endopeptidase was isolated from a Xanthomonas sp. and characterized with respect to physicochemical and enzymatic properties. The enzyme is composed of a single peptide chain with a molecular weight of 75,000. The isoelectric point is 6.2. It is inhibited by diisopropylfluorophosphate and may therefore be classified as a serine endopeptidase. The activity profile is bell shaped with an optimum at pH 7.5. By using synthetic peptide substrates and intramolecular fluorescence quenching it was possible to study the influence of substrate structure on the rate of hydrolysis. The enzyme specifically hydrolyzed Pro-X peptide bonds. With Glu at position X, low rates of hydrolysis were observed; otherwise the enzyme exhibited little preference for particular amino acid residues at position X. A similar substrate preference was observed with respect to the amino acid residue preceding the prolyl residue in the substrate. The enzyme required a minimum of two amino acid residues toward the N terminus from the scissile bond, but further elongation of the peptide chain by up to six amino acid residues caused only a threefold increase in the rate of hydrolysis. Attempts to cleave at the prolyl residues in oxidized RNase failed, indicating that the enzyme does not hydrolyze long peptides, a peculiar property it shares with other proline-specific endopeptidases.     

Substrate specificity of Ca(2+)/calmodulin-dependent protein kinase phosphatase: kinetic studies using synthetic phosphopeptides as model substrates

Ca(2+)/calmodulin-dependent protein kinase phosphatase (CaMKPase) dephosphorylates and regulates multifunctional Ca(2+)/calmodulin-dependent protein kinases. In order to elucidate the mechanism of substrate recognition by CaMKPase, we chemically synthesized a variety of phosphopeptide analogs and carried out kinetic analysis using them as CaMKPase substrates. This is the first report using systematically synthesized phosphopeptides as substrates for kinetic studies on substrate specificities of protein Ser/Thr phosphatases. CaMKPase was shown to be a protein Ser/Thr phosphatase having a strong preference for a phospho-Thr residue. A Pro residue adjacent to the dephosphorylation site on the C-terminal side and acidic clusters around the dephosphorylation site had detrimental effects on dephosphorylation by CaMKPase. Deletion analysis of a model substrate peptide revealed that the minimal length of the substrate peptide was only 2 to 3 amino acid residues including the dephosphorylation site. The residues on the C-terminal side of the dephosphorylation site were not essential for dephosphorylation, whereas the residue adjacent to the dephosphorylation site on the N-terminal side was essential. Ala-scanning analysis suggested that CaMKPase did not recognize a specific motif around the dephosphorylation site. Myosin light chain phosphorylated by protein kinase C and Erk2 phosphorylated by MEK1 were poor substrates for CaMKPase, while a synthetic phosphopeptide corresponding to the sequence around the phosphorylation site of the former was not dephosphorylated by CaMKPase but that of the latter was fairly good substrate. These data suggest that substrate specificity of CaMKPase is determined by higher-order structure of the substrate protein rather than by the primary structure around its dephosphorylation site. Use of phosphopeptide substrates also revealed that poly-L-lysine, an activator for CaMKPase, activated the enzyme mainly through increase in the V(max) values.     

New substrates for enteropeptidase. I. Biologically active hepta-nonapeptides

Enteropeptidase (enterokinase, EC 3.4.21.9) hydrolyzes peptide bonds formed by carboxyl groups of Lys or Arg residue provided that less than four negatively charged amino acid residues are in positions P2-P5 of its substrate. We determined the kinetic parameters of three substrates of this type: human angiotensin II (AT) (DR decreases VYIHPF) and the Hb(2-8) (LTAEEK decreases A) and Hb(1-9) (MLTAEEK decreases AA) peptides of the cattle hemoglobin beta-chain. The Km values for all the substrates (approximately 10(-3) M) were one order of magnitude higher than those of the typical synthetic substrates of enteropeptidase or chimeric proteins with the -DDDDK- full-size linker (Km approximately 10(-4) M). The kcat values for AT and Hb(2-8) were also close and low (approximately 30 min-1). The general hydrolysis efficiency of such substrates is no more than 1% of the corresponding value for the typical peptide and protein substrates of the enteropeptidase. However, the elongation of Hb(2-8) peptide by one amino acid residue from its N- or C-terminus results in a dramatic increase in the catalytic efficiency of the hydrolysis: the kcat value for Hb(1-9) is 1510 min-1, which means that it is hydrolyzed only three times less effective than the chimeric protein with the full-size linker.     

Mammalian chymotrypsin-like enzymes. Comparative reactivities of rat mast cell proteases, human and dog skin chymases, and human cathepsin G with peptide 4-nitroanilide substrates and with peptide chloromethyl ketone and sulfonyl fluoride inhibitors

The extended substrate binding sites of several chymotrypsin-like serine proteases, including rat mast cell proteases I and II (RMCP I and II, respectively) and human and dog skin chymases, have been investigated by using peptide 4-nitroanilide substrates. In general, these enzymes preferred a P1 Phe residue and hydrophobic amino acid residues in P2 and P3. A P2 Pro residue was also found to be quite acceptable. The S4 subsites of these enzymes are less restrictive than the other subsites investigated. The substrate specificity of these enzymes was also investigated by using substrates which contain model desmosine residues and peptides with amino acid sequences of the physiologically important substrates angiotensin I and angiotensinogen and alpha 1-antichymotrypsin, the major plasma inhibitor for chymotrypsin-like enzymes. These substrates were less reactive than the most reactive tripeptide reported here, Suc-Val-Pro-Phe-NA. The thiobenzyl ester Suc-Val-Pro-Phe-SBzl was found to be an extremely reactive substrate for the enzymes tested and was 6-171-fold more reactive than the 4-nitroanilide substrate. The four chymotrypsin-like enzymes were inhibited by chymostatin and N-substituted saccharin derivatives which had KI values in the micromolar range. In addition, several potent peptide chloromethyl ketone and substituted benzenesulfonyl fluoride irreversible inhibitors for these enzymes were discovered. The most potent sulfonyl fluoride inhibitor for RMCP I, RMCP II, and human skin chymase, 2-(Z-NHCH2CONH)C6H4SO2F, had kobsd/[I] values of 2500, 270, and 1800 M-1 s-1, respectively. The substrates and inhibitors reported here should be extremely useful in elucidating the physiological roles of these proteases.     

Anthranilamide and nitrotyrosine as a donor-acceptor pair in internally quenched fluorescent substrates for endopeptidases: multicolumn peptide synthesis of enzyme substrates for subtilisin Carlsberg and pepsin

The preparations of N alpha-Fmoc-3-nitro-L-tyrosine and N-Boc-anthranilic acid Dhbt ester and their application to parallel multiple column solid-phase peptide synthesis is described. A series of peptide substrates containing an anthraniloyl group at the amino terminus and a 3-nitrotyrosyl residue close to the carboxyl terminus have been synthesized. The fluorescence of the anthraniloyl group, intramolecularly quenched by the 3-nitrotyrosine, increases with cleavage of peptide bonds situated between the two groups. The quenching mechanism is of the long-range resonance energy transfer type and long peptide substrates were constructed and used for kinetic measurement on subtilisin Carlsberg and pepsin. Complete quenching was observed even with more than 20 A between the centers of the chromophores, and substrates with up to 50 A between the chromophores were synthesized. The importance of long substrates for optimal enzymatic activity was demonstrated.     

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.     

A systematic series of synthetic chromophoric substrates for aspartic proteinases.

The hydrolysis of the chromogenic peptide Pro-Thr-Glu-Phe-Phe(4-NO2)-Arg-Leu at the Phe-Phe(4-NO2) bond by nine aspartic proteinases of animal origin and seven enzymes from micro-organisms is described [Phe(4-NO2) is p-nitro-L-phenylalanine]. A further series of six peptides was synthesized in which the residue in the P3 position was systematically varied from hydrophobic to hydrophilic. The Phe-Phe(4-NO2) bond was established as the only peptide bond cleaved, and kinetic constants were obtained for the hydrolysis of these peptide substrates by a representative selection of aspartic proteinases of animal and microbial origin. The value of these water-soluble substrates for structure-function investigations is discussed.     

Biosynthesis of peptide neurotransmitters: studies on the formation of peptide amides

A high proportion of peptide transmitters and peptide hormones terminate their peptide chain in a C-terminal amide group which is essential for their biological activity. The specificity of an enzyme that catalyses the formation of the amide was investigated with the aid of synthetic peptide substrates. With peptides containing l-amino acids the enzyme exhibited an essential requirement for glycine in the C-terminal position; amidation did not take place with peptides that had leucine, alanine, glutamic acid, lysine or N-methylglycine at the C-terminus and a peptide extended by the attachment of lysine to the C-terminal glycine did not act as a substrate. Amidation did occur with a peptide containing C-terminal D-alanine but no reaction was detected with peptides having C-terminal, D-serine or D-leucine. In tripeptides with a neutral amino acid in the penultimate position, amidation, took place readily but the reaction was slower when this position was occupied by an acidic or a basic residue. A series of overlapping peptides with C-terminal glycine, based on partial sequences of calcitonin, underwent amidation at similar rates, indicating that the amidating enzyme recognizes only a limited sequence at the C-terminus of its substrates. The results provide evidence that the amidating enzyme has a highly compact substrate binding site.     

Substrate specificity of platypus venom L-to-D-peptide isomerase

The L-to-D-peptide isomerase from the venom of the platypus (Ornithorhyncus anatinus) is the first such enzyme to be reported for a mammal. In delineating its catalytic mechanism and broader roles in the animal, its substrate specificity was explored. We used N-terminal segments of defensin-like peptides DLP-2 and DLP-4 and natriuretic peptide OvCNP from the venom as substrates. The DLP analogues IMFsrs and ImFsrs (srs is a solubilizing chain; lowercase letters denote D-amino acid) were effective substrates for the isomerase; it appears to recognize the N-terminal tripeptide sequence Ile-Xaa-Phe-. A suite of 26 mutants of these hexapeptides was synthesized by replacing the second residue (Met) with another amino acid, viz. Ala, alpha-aminobutyric acid, Ile, Leu, Lys, norleucine, Phe, Tyr, and Val. It was shown that mutant peptides incorporating norleucine and Phe are substrates and exhibit L- or D-amino acid isomerization, but mutant peptides that contain residues with shorter, beta-branched or long side chains with polar terminal groups, viz. Ala, alpha-aminobutyric acid, Ile, Val, Leu, Lys, and Tyr, respectively, are not substrates. It was demonstrated that at least three N-terminal amino acid residues are absolutely essential for L-to-D-isomerization; furthermore, the third amino acid must be a Phe residue. None of the hexapeptides based on LLH, the first three residues of OvCNP, were substrates. A consistent 2-base mechanism is proposed for the isomerization; abstraction of a proton by 1 base is concomitant with delivery of a proton by the conjugate acid of a second base.     

Comparative study of various serine alkaline proteinases from microorganisms. Esterase activity against N-acylated peptide ester substrates

Hydrolyses of N-acylated peptide ester substrates by various serine alkaline proteinases from bacterial and mold origin were compared using Ac- or Z-(Ala)_m-X-OMe (m = 0-2 or 0-3; X = phenylalanine, alanine, and lysine) as esterase substrates. The results indicated that the esterase activities of these enzymes were markedly promoted by elongating the peptide chain from P₁ to P₂ or P₃ with alanine, irrespective of the kind of the amino acid residue at the P₁-position (amino acid residues in peptide substrates are numbered according to the system of Schechter and Berger (1)). The effect of the kind of amino acid residue at the P₂-position was further determined using Z-X-Lys-OMe (X = glycine, alanine, leucine, or phenylalanine) as esterase substrates. Alanine was the most efficient residue as X with subtilisins and Streptomyces fradiae Ib enzyme, while leucine or phenylalanine were most efficient with the enzymes from Streptomyces fradiae II, Aspergillus sojae, and Aspergillus melleus. All the serine alkaline proteinases tested in this study were sensitive to Z-Ala-Gly-PheCH₂Cl, the dependence of inhibition on the inhibitor concentration differed among the enzymes.     

COOH-terminal methylation of lamin B and inhibition of methylation by farnesylated peptides corresponding to lamin B and other CAAX motif proteins.

Previous reports from this laboratory have demonstrated that lamin B is reversibly methylesterified in a cell cycle-dependent manner. The site of this methylation, however, was not identified. In this report, we describe a single major methylated product obtained following reversed-phase high-performance liquid chromatographic analysis of peptides generated by proteolytic digestion of lamin B from rat liver nuclear envelopes. This peptide was retained on a lamin B COOH-terminal-specific antibody-affinity column, and COOH-terminal localization was confirmed by amino acid sequencing. Two other COOH-terminal peptides were found but were not methylated and differed in sequence by at least a single residue from the methylated peptide, indicating the existence of two lamin B gene products. Tetrapeptides, representing the putative mature COOH termini of lamin B, K-ras-2A, and unprocessed lamin A, were synthesized with or without farnesyl modification of the COOH-terminal cysteines. All three farnesylated peptides served as substrates for the partially purified lamin B methyltransferase with apparent Km values of 4.5, 0.69, and 21 microM, respectively. Nonfarnesylated peptides were not substrates for the enzyme. The three farnesylated peptides were also effective to varying degrees at inhibiting the methylation of lamin B and other cellular proteins in cell lysates.     

Glycine flanked by hydrophobic bulky amino acid residues as minimal sequence for effective subtilisin catalysis.

The specificity of alkaline mesentericopeptidase (a proteinase closely related to subtilisin BPN') for the C-terminal moiety of the peptide substrate (Pi' specificity) has been studied in both hydrolysis and aminolysis reactions. N-Anthraniloylated peptide p-nitroanilides as fluorogenic substrates and amino acid or peptide derivatives as nucleophiles were used in the enzymic peptide hydrolysis and synthesis. Both hydrolysis and aminolysis kinetic data suggest a stringent specificity of mesentericopeptidase and related subtilisins to glycine as P1' residue and predilection for bulky hydrophobic P2' residues. A synergism in the action of S1' and S2'subsites has been observed. It appears that glycine flanked on both sides by hydrophobic bulky amino acid residues is the minimal amino acid sequence for an effective subtilisin catalysis.     

Studies on the specificity of acetylaminoacyl-peptide hydrolase.

In a continuing attempt to explore the types of specificity determinants that may affect protein-protein (peptide) interactions, a number of short (2-5 residues) acetylated peptides have been compared as substrates for the enzyme acetylaminoacyl-peptide hydrolase (EC 3.4.19.1). The reference substrate was Ac-AAAA, and most of the other substrates were derived from this basic structure by single amino acid substitutions. The Km and kcat for the different substrates were determined by standard steady-state kinetics, and the corresponding delta delta GT++ value derived from kcat/Km was used for the comparison, setting delta detal GT++ for Ac-AAAA equal to 0. The best substrates were found to be those containing negative charges (Asp > Glu) or aromatic residues in positions 1', 2', or 3' (delta delta GT++ values of 2-5 kJ); the negative charge provided by the C-terminus of the substrate also appears to be important, since the amide and O-Me ester derivatives caused a change in delta delta GT++ values of -7 to -8 kJ from the reference peptide. The stimulating effect of the negative charges is consistent with the inhibitory effect of positive charges in similar peptides (Krishna RG, Wold F, 1992, Protein Sci 1:582-589), and the proposed active site model incorporates subsites for both charge-charge and hydrophobic interactions. In assessing all the data, it is clear that the properties of the individual substrates reflect the total make-up of each peptide and not only the effect of a single residue in a given position.(ABSTRACT TRUNCATED AT 250 WORDS)     

A synthetic peptide derived from the non-structural protein 3 of hepatitis C virus serves as a specific substrate for PKC

A synthetic peptide corresponding to the amino acid sequence Arg1487-Arg-Gly-Arg-Thr-Gly-Arg-Gly-Arg-Arg-Gly-Ile-Tyr-Arg1500 of the hepatitis C virus (HCV) polyprotein was found to be a selective substrate for protein kinase C (PKC). In the presence of Ca2+, TPA and phospholipid, PKC phosphorylates the peptide [termed HCV(1487-1500)] with a Km of 11 microM and Vmax of 24 micromol x min(-1) x mg(-1). HCV(1487-1500) acts as a competitive inhibitor of PKC towards other peptide or protein substrates and inhibits the kinase activity with an IC50 corresponding to the Km values measured for the substrates. N- or C-terminally deleted analogs of HCV(1487-1500) did not show inhibitory effects and were only marginally or not phosphorylatable. We designed an additional peptide in which the tyrosine residue was replaced by phenylalanine ([Phe1499]HCV(1487-1500)). This peptide was neither phosphorylated by other serine/threonine kinases tested nor by whole cell extracts prepared from PKC-depleted cells. [Phe1499]HCV(1487-1500) was used to monitor the TPA-induced translocation of PKC activity to the particulate fraction in JB6 cells. The use of SDS/PAGE to separate the peptide from ATP and Pi allowed to monitor simultaneously PKC autophosphorylation and phosphorylation of the peptide. The data presented here show that[Phe1499]HCV(1487-1500) can serve as a convenient tool for investigations of PKC activity also in the presence of other kinases in tissues or in crude cell extracts.     

Effects of secondary interactions on the kinetics of peptide and peptide ester hydrolysis by tissue kallikrein and trypsin

Kinetic constants for the hydrolysis by porcine tissue beta-kallikrein B and by bovine trypsin of a number of peptides related to the sequence of kininogen (also one containing a P2 glycine residue instead of phenylalanine) and of a series of corresponding arginyl peptide esters with various apolar P2 residues have been determined under strictly comparative conditions. kcat and kcat/Km values for the hydrolysis of the Arg-Ser bonds of the peptides by trypsin are conspicuously high. kcat for the best of the peptide substrates, Ac-Phe-Arg-Ser-Val-NH2, even reaches kcat for the corresponding methyl ester, indicating rate-limiting deacylation also in the hydrolysis of a peptide bond by this enzyme. kcat/Km for the hydrolysis of the peptide esters with different nonpolar L-amino acids in P2 is remarkably constant (range 1.7), as it is for the pair of the above pentapeptides with P2 glycine or phenylalanine. kcat for the ester substrates varies fivefold, however, being greatest for the P2 glycine compounds. Obviously, an increased potential of a P2 residue for interactions with the enzyme lowers the rate of deacylation. In contrast to results obtained with chymotrypsin and pancreatic elastase, trypsin is well able to tolerate a P3 proline residue. In the hydrolysis of peptide esters, tissue kallikrein is definitely superior to trypsin. Conversely, peptide bonds are hydrolyzed less efficiently by tissue kallikrein and the acylation reaction is rate-limiting. The influence of the length of peptide substrates is similar in both enzymes and indicates an extension of the substrate recognition site from subsite S3 to at least S'3 of tissue kallikrein and the importance of a hydrogen bond between the P3 carbonyl group and Gly-216 of the enzymes. Tissue kallikrein also tolerates a P3 proline residue well. In sharp contrast to the behaviour of trypsin is the very strong influence of the P2 residue in tissue-kallikrein-catalyzed reactions. kcat/Km varies 75-fold in the series of the dipeptide esters with nonpolar L-amino acid residues in P2, a P2 glycine residue furnishing the worst and phenylalanine the best substrate, whereas this exchange in the pentapeptides changes kcat/Km as much as 730-fold. This behaviour, together with the high value of kcat/Km for Ac-Phe-Arg-OMe of 3.75 X 10(7) M-1 s-1, suggests rate-limiting binding (k1) in the hydrolysis of the best ester substrates.(ABSTRACT TRUNCATED AT 400 WORDS)     

The stereochemistry of hydrogen abstraction in vitamin K-dependent carboxylation

The stereochemistry of the hydrogen abstraction in the vitamin K-dependent carboxylation of synthetic peptides has been investigated; the carboxylation rates of various peptidic substrates containing a stereospecifically 4-monodeuterated glutamic acid residue have been compared to that of nondeuterated peptides. A significant isotope effect was found only with the substrates containing (4S)-4-deuterated glutamic acid. These data reveal that the rat liver microsomal vitamin K-dependent carboxylase acts stereospecifically in abstracting the 4-pro-S hydrogen of the glutamyl residue. The low values of the measured isotope effects indicate that the hydrogen abstraction does not constitute a limiting step in the carboxylation mechanism.     

Substrate specificities and inhibition of two hemorrhagic zinc proteases Ht-c and Ht-d from Crotalus atrox venom

The proteolytic specificities of two zinc hemorrhagic toxins (Ht-c and Ht-d), isolated from Crotalus atrox venom, were investigated by using the oxidized B chain of bovine insulin and synthetic peptide substrates. The enzymes cleaved the Ala14-Leu15 bond of the insulin B chain most rapidly and the Tyr16-Leu17 slightly more slowly. The His5-Leu6, His10-Leu11, and Gly23-Phe24 bonds were also cleaved but at considerably slower rates. In order to assess the substrate length preferences of the enzymes, peptide analogs of the B chain about the Ala14-Leu15 bond were synthesized ranging in length from four to seven residues. The heptapeptide NH2-Leu-Val-Glu-Ala-Leu-Tyr-Leu-COOH was the best peptide substrate tested with the other peptides having decreasing kcat/Km values with decreasing length. The tetrapeptide NH2-Ala-Leu-Tyr-Leu-COOH was not cleaved by the enzymes. Furthermore, this peptide was shown to serve as a competitive inhibitor of the toxins. The N-acetylated pentapeptides and hexapeptides, synthesized to probe the active site environment of the enzymes, were significantly better substrates than their unacetylated counterparts. The toxins had the highest kcat/Km values for the acetylated peptide Ac-Val-Ala-Leu-Leu-Ala-COOH. The data suggest that the toxins may indeed have extended substrate-binding sites, which may accommodate at least six amino acid residues. The best substrate examined thus far for the toxins is the fluorogenic peptide analog 2-aminobenzoyl-Ala-Gly-Leu-Ala-4-nitrobenzylamide, suggestive of similarities between the toxins and mammalian collagenases as well as thermolysin. Mechanisms for inhibition of the enzymes were investigated using amino acid hydroxamates, chloromethyl esters, phosphoramidon and the peptide NH2-Ala-Leu-Tyr-Leu-COOH. All of these inhibitors had Ki values in the 10(-4) M range.     

Effects of amino acid sequence, buffers, and ionic strength on the rate and mechanism of deamidation of asparagine residues in small peptides

The nonenzymatic rates of deamidation of Asn residues in a series of pentapeptides with the sequences VSNXV and VXNSV, where X is one of 10 different amino acids, were determined at neutral, alkaline, and acid pH values. The results demonstrate that in neutral and alkaline solutions the amino acid residue on the amino side of the Asn had little or no effect on the rate of deamidation regardless of its charge or size. The group on the carboxyl side of Asn affected the rate of deamidation significantly. Increasing size and branching in the side chain of this residue decreased the rate of deamidation by as much as 70-fold compared to glycine in the N-G sequence, which had the greatest rate of deamidation. In acidic solution, the rate of deamidation of the Asn residue was not affected by the amino acid sequence of the peptide. The products for each deamidation reaction were tested for the formation of isoAsp residues. In neutral and alkaline solutions, all products showed that the isoAsp:Asp peptide products were formed in about a 3:1 ratio. In acidic solution, the Asp peptide was the only deamidation product formed. All peptides in which a Ser residue follows the Asn residue were found to undergo a peptide cleavage reaction in neutral and alkaline solutions, yielding a tripeptide and a dipeptide. The rate of the cleavage reaction was about 10% of the rate of the deamidation pathway at neutral and alkaline pH values. The rates of deamidation of Asn residues in the peptides studied were not affected by ionic strength, and were not specific base catalyzed. General base catalysis was observed for small bases like ammonia. A model for the deamidation reaction is proposed to account for the observed effects.