Acyl and amino intermediates in reactions catalyzed by thermolysin

Thermolysin showed peculiar transpeptidation reactions. Leu-Leu and/or Leu-Leu-Leu were produced at $\text{ca}$. pH 7 from Leu-Leu-NH₂ and Cbz-Leu-Leu. Isotope experiments indicated that the transpeptidation products did not use leucine released from the substrates as an acceptor. With Leu-Trp-Met, Leu-Leu, Leu-Leu-Leu and Met-Met were produced as transpeptidation products. A comparative study was done with α-chymotrypsin and pepsin. These results would indicate that thermolysin catalyzed reactions proceed via both acyl and amino intermediates depending upon the substrates, which has been proposed for the mechanism of pepsin. This may also be true in some cases for chymotrypsin and other proteases, which have been known as enzymes of the acyl-enzyme mechanism.     

Acyl intermediates in penicillopepsin-catalysed reactions and a discussion of the mechanism of action of pepsins.

Penicillopepsin catalyses transpeptidation reactions involving the transfer of the N-terminal amino acids of suitable substrates via covalent acyl intermediates to acceptor peptides, usually the substrate. The major products obtained when Phe-Tyr-Thr-Pro-Lys-Ala and Met-Leu-Gly were used as substrates were Phe-Phe and Met-Met respectively. With Met-Leu-Gly the tetrapeptide Met-Met-Leu-Gly was observed as probable intermediate. Co-incubation of Leu-Tyr-Leu and Phe-Tyr-Thr-Pro-Lys-Ala led to the formation of Leu-Phe and Phe-Leu as well as Leu-Leu and Phe-Phe. No reaction was observed with tripeptides in which the first or second amino acid is glycine. It appears that two amino aicds with large hydrophobic residues are needed for the transpeptidation reaction. Nucleophilic compounds other than peptides, such as hydroxylamine, aliphatic alcohols and dinitrophenylhydrazine, were not acceptors for the acyl group. Leucine, phenylalanine and leucine methyl ester also had no effect on the reaction. The transpeptidation reaction proceeded readily at pH 3.6 and 4.7. At pH 6.0 the reaction was slow and at pH 1.9 little or no transpeptidation was observed. Porcine pepsin catalyses similar transpeptidation reactions. Sequence studies show that porcine pepsin and penicillopepsin are homologous. The present study also suggests that they have a very similar mechanism. Evidence available at this time indicates that the mechanism of these enzymes is complex and may be modulated by secondary substrate-enzyme interactions. A hypothesis is presented which proposes that pepsin-catalysed reactions proceed via different covalent intermediates (amino-intermediates or acylintermediates) depending on the nature of the substrate. The possibility that some reactions do not involve covalent intermediates is also discussed.     

The specificity of some pig and human pepsins towards synthetic peptide substrates.

1. The peptidase activities of pig pepsins A and C and human pepsin and gastricsin were compared. 2. The peptides studied had the general formula A Leu Val-His-B. Hydrolysis at 37 degrees C and pH 2.07 occurred at the amino side of the leucine residue for all the enzymes and all the peptides. 3. When A was Ac-Ala the peptides were hydrolysed under these conditions slowly by pig pepsin C only. 4. Pig pepsin A and human pepsin were unable to hydrolyse the tyrosine-containing peptides under the conditions tested. Gastricsin (human pepsin C) had about one-third of the activity of pig pepsin C with these substrates. 5. The increase in the rate of hydrolysis caused by the extension of the chain by a single alanine residue was most marked for pig pepsin A and human pepsin.     

Surprising consequences of the tendency of pepsin to catalyze condensation reactions between small peptides

Pepsin catalyzes numerous acyl-transfer reactions. Are peptic acyl-enzyme intermediates involved in such reactions? To start, we examine the cleavage of Leu-Trp-Met-Arg at pH 3.4-4.5 in the presence of 25 mM tryptophanamide. Substantial amounts of Leu-TrpNH2 are generated. However, the appearance of this acyl-transfer product cannot be attributed to the intervention of Leu-pepsin and its trapping by tryptophanamide. Experiment proves that Leu-Trp-Met-Arg affords Leu3, which, in turn, reacts with tryptophanamide to produce Leu-TrpNH2. Both the formation of Leu3 from Leu-Trp-Met-Arg and the conversion of Leu3 + tryptophanamide into Leu-TrpNH2 can potentially implicate the generation and trapping of a Leu-pepsin intermediate. Does experiment support either possibility? The answer is no. Our data show that most of the Leu3 derived from Leu-Trp-Met-Arg stems from an autocatalytic condensation process whereby Leu3 already present speeds the conversion of the leucine residues of unreacted Leu-Trp-Met-Arg into more Leu3. Technical problems have prevented us from determining whether the first Leu3 formed results from the trapping of an acyl-enzyme intermediate. The generation of Leu-TrpNH2 from Leu3 was studied primarily via a more tractable analogous reaction: Leu-Trp-Leu + tryptophanamide leads to Leu-TrpNH2. The mechanism governing these transformations is highly complex. Its major feature is an initial condensation between two molecules of substrate. All the examples investigated further illustrate the marked tendency of pepsin to catalyze condensation reactions between suitably constructed small peptides. The prevalence of these reactions complicates the interpretation of much data bearing on pepsin's mechanism of action.     

Transpeptidation reactions of porcine pepsin. Formation of tetrapeptides from dipeptide substrates

Pepsin-catalyzed transpeptidation was studied by high resolution 75 MHz 13C nuclear magnetic resonance spectroscopy. Enrichment with 13C at the carbonyl carbons of the substrates Leu-Tyr-NH2 and Leu-Leu-NH2 facilitated detection and identification of the transpeptidation and hydrolysis products of enzymic action. Porcine pepsin was found in each case to synthesize and release the tetrapeptide Leu-Leu-Leu-Leu as the primary product of transpeptidation, the longest oligomeric product of transpeptidation observed to date. Productive binding of the dipeptide substrates into the active site groove of pepsin required an induction period of several minutes. Quenching experiments suggested the presence of strongly bound intermediate forms of Leu and Leu-Leu prior to observation of any enzyme-free products. The finding of the tetrapeptide as a primary product is discussed as an instance where transpeptidation of the tripeptide competes successfully with the action of pepsin subsite S3 as a trigger for product release.     

Kinetic studies of sheep kidney gamma-glutamyl transpeptidase.

The kinetics of sheep kidney gamma-glutamyl transpeptidase was studied using a novel substrate L-alpha-methyl-gamma-glutamyl-L-alpha-aminobutyrate. When the substrate was incubated with the enzyme in the presence of an amino acid or peptide acceptor, the corresponding L-alpha-methyl-gamma-glutamyl derivatives of the acceptors were formed. In the absence of acceptor only hydrolysis occurred, and no transpeptidation products were detected. The presence of the methyl group on the alpha-carbon apparently prevents enzymatic transfer of the L-alpha-methyl-gamma-glutamyl residue to the amino group of the substrate itself (autotranspeptidation). When the enzyme was incubated with conventional substrates, such as glutathione or gamma-glutamyl-p-nitroanilide and an amino acid acceptor, hydrolysis, autotranspeptidation, and transpeptidation to the acceptor occurred concurrently. Initial velocity measurements in which the concentration of L-alpha-methyl-gamma-glutamyl-L-alpha-aminobutyrate was varied at several fixed acceptor concentrations, and either the release of alpha-aminobutyrate or the formation of the transpeptidation products was determined, yielded results which are consistent with a ping-pong mechanism modified by a hydrolytic shunt. A scheme of such a mechanism is presented. This mechanism predicts the formation of an alpha-methyl-gamma-glutamyl-enzyme intermediate, which can react with an amino acid to form the transpeptidation product; or in the absence of, or in the presence of low concentrations of amino acids, can react with water to form the hydrolytic products. Kinetic derivations for the reaction of the enzyme with the conventional substrate gamma-glutamyl-p-nitroanilide predict either linear or nonlinear double-reciprocal plots, depending on the prevalence of the hydrolytic, autotranspeptidation, or transpeptidation reactions. The results of kinetic experiments confirmed these predictions.     

Development of an HPLC assay for Staphylococcus aureus sortase: evidence for the formation of the kinetically competent acyl enzyme intermediate

Many bacterial surface proteins containing an LPXTG motif are anchored to the cell wall peptidoglycan by catalysis with the thiol transpeptidase sortase. The transpeptidation and hydrolysis reactions of sortase have been proposed to proceed through a common acyl enzyme intermediate. The reactions of Staphylococcus aureus sortase with fluorogenic substrate Abz-LPETG-Dnp in the presence or absence of triglycine were characterized in this study to gain additional insight into the kinetic mechanism of sortase. We report here the development of a reverse-phase HPLC assay to identify and characterize sortase reaction intermediates. The HPLC results provide for the first time clear evidence for the formation of a kinetically competent acyl enzyme intermediate during the overall transpeptidation reaction. The results also suggest that sortase undergoes an unexpected intramolecular acyl transfer reaction in the absence of a nucleophile. The significance of this type of HPLC assay as a tool to study enzyme mechanism is discussed.     

Transpeptidation reactions of a specific substrate catalyzed by the Streptomyces R61 DD-peptidase: the structural basis of acyl acceptor specificity

Bacterial dd-peptidases, the targets of beta-lactam antibiotics, are believed to catalyze d-alanyl-d-alanine carboxypeptidase and transpeptidase reactions in vivo. To date, however, there have been few concerted attempts to explore the kinetic and thermodynamic specificities of the active sites of these enzymes. We have shown that the peptidoglycan-mimetic peptide, glycyl-l-alpha-amino-epsilon-pimelyl-d-alanyl-d-alanine, 1, is a very specific and reactive carboxypeptidase substrate of the Streptomyces R61 dd-peptidase [Anderson, J. W., and Pratt, R. F. (2000) Biochemistry 39, 12200-12209]. In the present paper, we explore the transpeptidation reactions of this substrate, where the enzyme catalyzes transfer of the glycyl-l-alpha-amino-epsilon-pimelyl-d-alanyl moiety to amines. These reactions are believed to occur through capture of an acyl-enzyme intermediate by amines rather than water. Experiments show that effective acyl acceptors require a carboxylate group and thus are amino acids and peptides. d(but not l)-amino acids, analogues of the leaving group of 1, are good acceptors. The effectiveness of d-alanine as an acceptor increases with pH, suggesting that the bound and reactive form of an amino acid acceptor is the free amine. Certain glycyl-l(but not d)-amino acids, such as glycyl-l-alanine and glycyl-l-phenylalanine, are also good acceptors. These molecules may resemble the N-terminus of the Streptomyces stem peptides that, presumably, are the acceptors in vivo. The acyl acceptor binding site therefore demonstrates a dual specificity. That d-alanyl-l-alanine shows little activity as an acceptor suggested that, on binding of acceptors to the enzyme, the carboxylate of d-amino acids does not overlap with the peptide carbonyl group of glycyl-l-amino acids. Molecular modeling of transpeptidation tetrahedral intermediates and products demonstrated the likely structural bases for the stereospecificity of the acceptors and the nature of the dual function acceptor binding site. For both groups of acceptors, the terminal carboxylate appeared to be anchored at the active site by interaction with Arg 285 and Thr 299.     

High-efficiency transpeptidation catalysed by clostripain and electrostatic effects in substrate specificity.

Clostripain catalyses the transpeptidation between benzoylarginin ethyl ester and amino acid amides, oligopeptides, insulin A- and B-chains and tryptic peptides of myoglobin at millimolar substrate concentrations. The reactions proceed with temporary accumulation of the products, followed by hydrolytic decomposition. The yield was not affected significantly by the type of N-terminal amino acid, but was diminished markedly by the negative charges of the amine components. The yields for natural peptides were linearly related to the charge density of the peptides.     

Pepsin as a catalyst of peptide synthesis. Enzyme co-precipitation with emerging peptide products.

Pepsin successfully catalyzed the synthesis of several peptide derivatives from N-protected di- or tripeptides and amino acid or peptide esters or p-nitroanilides in dimethylformamide-water solutions at pH 4.6. An optimal substrates:pepsin ratio depended on the structure of starting peptides, especially their fit to the substrate binding sites of the enzyme. For hexapeptide Z-Ala-Ala-Phe-Leu-Ala-Ala-OCH3 formation, an equilibrium yield was attained at 1:3.10(5) enzyme-substrates ratio that indicated high efficiency of pepsin in synthesis reactions. In the course of the equilibrium peptide synthesis, pepsin gradually disappeared from the liquid phase due to its entrapment within a gel, formed by the hexapeptide product, while retaining its activity. The inclusion into the precipitate was not specific for pepsin, so far as inert proteins, lysozyme, ribonuclease A and carbonic anhydrase, when added to the reaction mixture, became also co-precipitated with the hexapeptide formed. It appears that co-precipitation of pepsin, an important factor limiting the enzyme efficiency, might be operative as well for other proteinases used to catalyze peptide synthesis.     

Large peptides of bovine and guinea pig myelin basic proteins produced by limited peptic hydrolysis.

Bovine and guinea pig myelin basic proteins were cleaved with pepsin at pH 3.0 or pH 6.0 (enzyme/substrate, 1:500, w/w), and the peptides were isolated and identified. At pH 3.0 cleavage of the bovine protein occurred principally at three sites: Phe-Phe (88-89), Phe-Phe (42-43), and Leu-Asp (36-37). Minor cleavages occurred at Leu-Ser (110-111), Phe-Ser (113-114), and Ile-Phe (152-153). A study of the time course of the hydrolysis showed that the reaction was biphasic; nearly all of the protein was cleaved at Phe-Phe (88-89) before significant cleavages at other sites occurred. At pH 6.0 cleavage of the bovine protein occurred almost exclusively at a single site, the Phe-Phe bond at position 88-89, resulting in bisection of the protein. Treatment of the guinea pig protein with pepsin under the same conditions resulted in the production of peptides which were identical with those of the bovine protein in chromatographic and electrophoretic properties and in N-terminal and C-terminal residues but which differed slightly in amino acid composition.     

Specificity and reversibility of the transpeptidation reaction catalyzed by the Streptomyces R61 D-Ala-D-Ala peptidase

The specificity of the Streptomyces R61 penicillin-sensitive D-Ala-D-Ala peptidase has been re-examined with the help of synthetic substrates. The products of the transpeptidation reactions obtained with Gly-L-Xaa dipeptides as acceptor substrates are themselves poor substrates of the enzyme. This is in apparent contradiction with the classically accepted specificity rules for D-Ala-D-Ala peptidases. The Gly-L-Xaa dipeptide is regenerated by both the hydrolysis and transpeptidation reactions. The latter reaction is observed when another Gly-L-Xaa peptide or D-Alanine are supplied as acceptors. Utilization of substrates in which the terminal -COO(-) group has been esterified or amidated shows that a free carboxylate is not an absolute prerequisite for activity. The results are discussed in the context of the expected reversibility of the transpeptidation reaction.     

Structural requirements for nucleophilic substrates of carboxypeptidase Y

The composition and structural aspects of the amino and carboxylic acid groups required for incorporation into peptides by transpeptidation and inhibition of hydrolysis in carboxypeptidase Y-catalyzed reactions were studied. Separation of these two groups by even one carbon prevents incorporation by transpeptidation and does not inhibit incorporation of other amino acids into model peptides. Substitution of phosphonic or sulfonic acids for the carboxylic acid group also results in loss of incorporation by transpeptidation. Only the sulfonic acid analog of glycine causes inhibition of hydrolysis and this inhibition is lost when serine is included in the reaction. d-Serine is not incorporated by carboxypeptidase Y, and its presence in the reaction mixture does not inhibit the incorporation of the L-isomer.     

The reaction of tertiary amino alcohols with active esters: I. Acylation and deacylation steps

The reactions of triethanolamine and four other tertiary amino alcohols with six active ester substrates were studied in the pH range 6–10 at 30°C. The reaction products were in all cases the respective O-acyl-amino alcohols. Analysis of the effects of substituents in the leaving group as well as in the acyl moiety of the substrates showed that the ester product was formed by direct attack of the nucleophilic hydroxyl group. Comparison with reactions of tertiary amines with the same substrates supports this conclusion. The reactions of tertiary amino alcohols were also compared with those of zwitterionic quaternary amino alcohols and 3-quinuclidinol, a “rigid” tertiary amino alcohol. On the basis of these comparisons, it is proposed that one of the pathways for the predominant effect of the neutral species of tertiary amino alcohols involves intramolecular general base assistance by the tertiary amino group to the nucleophilic attack of the hydroxylic oxygen on the substrate. The contribution of this pathway to the rate of reaction is evaluated.In several systems the first product of the reaction, an O-acyl-amino alcohol, undergoes relatively rapid deacylation, the overall reaction being thus hydrolysis of active esters, catalyzed by the amino alcohol via an acylation-deacylation mechanism.     

Evidence that transpeptidation is a significant function of gamma-glutamyl transpeptidase

gamma-Glutamyl transpeptidase (purified from rat kidney) was incubated with glutathione and a mixture of amino acids that closely approximates the amino acid composition of blood plasma, and the relative extents of transpeptidation and hydrolysis were determined by quantitative measurement of the products formed (glutamate, cysteinylglycine, gamma-glutamyl amino acids). At pH 7.4, in the presence of 50 microM glutathione and the amino acid mixture, about 50% of the glutathione that was utilized participated in transpeptidation. Studies in which the formation of individual gamma-glutamyl amino acids was determined in the presence of glutathione and the amino acid mixture showed that L-cystine and L-glutamine are the most active amino acid acceptors, and that other neutral amino acids also participate in transpeptidation to a significant extent. These in vitro experiments are consistent with a number of other findings which indicate that transpeptidation is a significant physiological function of gamma-glutamyl transpeptidase.     

A mutant Bacillus subtilis gamma-glutamyltranspeptidase specialized in hydrolysis activity

gamma-Glutamyltranspeptidase (GGT) catalyzes the hydrolysis of gamma-glutamyl compounds and the transfer of their gamma-glutamyl moieties to amino acids and peptides. The transpeptidation activity of Bacillus subtilis GGT is about 10-fold higher than its hydrolysis activity. In B. subtilis GGT, substitution of Asp-445 with Ala abolished its transpeptidation activity. The specific activity for hydrolysis of D445A GGT was 40.2% of that of the wild-type GGT. The K(m) value for L-glutamine was 15.3 mM. D445A GGT was salt tolerant like the wild-type GGT. These results indicate that D445A GGT will be highly useful as a 'glutaminase' in food industry.     

The kinetics of hydrolysis of some synthetic substrates containing neutral hydrophilic groups by pig pepsin and chicken liver cathepsin D.

1. Several peptides containing either of the sequences -Phe(NO2)-Trp- and -Phe(NO2)-Phe- and an uncharged hydrophilic group were synthesized, and the steady-state kinetics of their hydrolysis by pig pepsin (EC 3.4.23.1) and chicken liver cathepsin D (EC 3.4.23.5) were determined. Despite the presence of a hydrophilic group to increase substrate solubility, it was not possible to achieve the condition [S]0 much greater than Km, and, in some cases, only values of kcat./Km could be determined by measuring the first-order rate constant when [S]0 much less than Km. 2. Occupancy of the P2 and P3 sites considerably enhanced the specificity constant, and alanine was more effective than glycine at site P2. 3. The specificity constants for the hydrolysis by pepsin of those substrates in the present series that contain an amino acid residue at site P3 are considerably lower than for comparable substrates containing a cationic group. This difference does not apply to cathepsin D. 4. Hydrolyses with cathepsin D commonly exhibited a lag phase, and a possible explanation for this is given.     

BRAIN PEPTIDASES: CONVERSION AND INACTIVATION OF KININ HORMONES

Abstract— Two enzymes that selectively hydrolyse kinins at pH 7.5 were obtained in partially purified form from the supernatant fraction of homogenates of previously frozen rabbit brain by gel filtration on Sephadex G-100. The enzymes were detected and their activity estimated by bioassay with the isolated guinea pig ileum The products of the enzymic reactions were identified by high voltage electrophoresis at pH 3.5 and by the determination with the amino acid analyser of the amino acids released from the kinins.
One enzyme, kinin-converting enzyme, catalyses the hydrolysis of kinin-10 (Lysbradykinin) and kinin-11 (Met-Lys-bradykinin) into kinin-9 (bradykinin). It also hydrolyses the aminoacyl-8-naphthylamides of methionine, lysine, arginine and leucine. The conversion of kinin-10 to kinin-9 was inhibited by puromycin (K_i 3.5 × 10⁻⁵ M) These properties are similar to those of brain arylamidases described in the literature.
Kininase, the second enzyme, inactives kinins 9, 10 and 11 by peptide-bond hydrolysis. Similar rates of release of arginine and phenylalanine were observed for the three kinins, suggesting that kininase acts at the carboxy-terminus of these peptides.
Our results suggest that brain contains proteases which apparently selectively metabolize polypeptide hormones that exert definite pharmacological effects on the central and peripheral nervous systems.     

The inhibition of pepsin-catalysed reactions by products and product analogues. Kinetic evidence for ordered release of products

1. The inhibition of pepsin-catalysed hydrolysis of N-acetyl-l-phenylalanyl-l-phenylalanylglycine by products and product analogues was studied. 2. The non-competitive nature of the inhibition by the product N-acetyl-l-phenylalanine confirms an ordered release of products, and points to a common mechanism (involving an amino-enzyme) for pepsin-catalysed transpeptidation and hydrolysis reactions. 3. N-Acetyl-l-phenylalanine ethyl ester is also a non-competitive inhibitor, but here the inhibition is of the ;dead-end' type. No ethanol is detectable in reaction mixtures, indicating that this ester cannot act as an amino group acceptor in a transpeptidation process. 4. The same is true for N-methanesulphonyl-l-phenylalanine methyl and methyl thiol esters. No methanethiol is liberated when the methyl thiol ester is present as an inhibitor of the hydrolytic reaction, and the hope that such a thiol ester would effectively trap the amino-enzyme was not fulfilled.     

Oligo(methionyl) proteins. Enzymatic hydrolysis of the model isopeptides N epsilon-oligo(L-methionyl)-L-lysine

A number of model isopeptides containing oligo(methionine) chains varying in length (2-5 residues) covalently linked to the epsilon-amino group of lysine were synthesized by solid-phase procedures. Hydrolysis of these peptides by pepsin, chymotrypsin, cathepsin C (dipeptidyl peptidase IV) and intestinal aminopeptidase N was investigated using high-performance liquid chromatography to identify and quantify the hydrolysis products. Methionine oligomers grafted onto lysine were cleaved to tripeptides by pepsin. Chymotrypsin preferentially hydrolyzed the methionyl-methionine bond preceding the isopeptide bond. Cathepsin C released dimethionyl units from the covalently attached polymers. Intestinal aminopeptidase caused efficient hydrolysis of both peptides and isopeptide bonds although free methionine decreased the cleavage of the latter bond. Hydrophobic characteristics of oligo(methionine) chains promoted enzyme-catalyzed transpeptidations resulting probably from acyl-transfer-type reactions. Complementary hydrolysis of the isopeptides by these digestive enzymes suggests that covalent attachment of oligo(amino acid)s to food proteins may improve their nutritional value.