Kinetic evidence for an acyl-enzyme intermediate in D-alanine carboxypeptidases of Bacillus subtilis and Bacillus stearothermophilus.

The kinetics of hydrolysis and transpeptidation of the synthetic substrate diacetyl-L-lysyl-D-alanyl-D-alanine and of the natural substrate UDP-acetylmuramyl pentapeptide and related compounds catalyzed by the D-alanine carboxypeptidases of Bacillus subtilis and Bacillus stearothermophilus in the presence of the nucleophiles hydroxylamine or glycine have been examined. These kinetic data suggest that an acyl-enzyme intermediate is formed in the first step of the reaction and that the transpeptidation is the consequence of the partitioning of this intermediate between water and the nucleophile in the second step.     

Enzymatic Modification of Proteins in Foodstuffs

Peptic hydrolyzate of soy protein was submitted to the plastein reaction with α-chymotrypsin under the following condition: substrate concentration, 20%; enzyme-substrate ratio by weight, 1/100; reaction pH, 7.0; and reaction temperature, 37°C. The plastein yield resulting from the plastein reaction for 24 hr was found to depend on the degree of hydrolysis of the substrate (per cent ratio between nitrogen amount in 10% trichloroacetic acid soluble nitrogen and that in whole hydrolyzate); the optimum degree of hydrolysis for the highest plastein yield seemed to lie around 80%. A turbidity appeared in the process of the plastein reaction, whose intensity was correlative to the plastein yield. The peptic hydrolyzate of soy protein per se had bitterness and its magnitude decreased with increasing plastein yield.

As a result of the plastein reaction applied for 24 hr to the hydrolyzate whose degree of hydrolysis was 80%, the average molecular weight estimated by the change in amino nitrogen content increased by approximately three times. The molecular weight distribution pattern obtained by gel filtration supported the above result. The total amount of amino acids liberated from the plastein reaction product by its treatment with either leucine aminopeptidase or carboxypeptidase A was significantly less than that liberated from the original hydrolyzate by its similar treatment. This result also supports the formation of higher-molecular protein-like substances by the plastein reaction. Deuteration study followed by IR spectrometry showed the occurrence of peptide bond formation, i.e. decrease in ionized carboxyl group at 1575 cm^?1 and increase in deuterated amide at 1450 cm^?1, even at the earlier stages of the plastein reaction.     

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.     

Kinetics of alpha-chymotrypsin catalyzed hydrolysis in equilibrium. II. Comparison of ester and amide substrates

Kinetic of the alpha-chymotrypsin catalyzed reversible hydrolytic reaction of methyl N-acetyl-L-phenylalaninate and N-acetyl-L-phenylalanylglycinamide at pH 5.5 and equilibrium conditions has been studied. The rates of the labeled reaction products incorporated into the substrate a different methanol concentrations shows that the reaction proceeds by a compulsory mechanism with the formation of N-acetyl-L-phenylalanine-alpha-chymotrypsin complex. For the amide substrate the data obtained are also in agreement with the compulsory mechanism of its hydrolysis. Equilibrium kinetics of ester and amide substrates hydrolysis has been compared.     

Enzymic properties of nitrated alpha-chymotrypsin and delta-chymotrypsin.

Chymotrypsinogen A and alpha-chymotrypsin are both nitrated at tyrosines 146 and 171 by reaction with tetranitromethane. This substitution was essentially without influence on the overall rate constant for hydrolyses of N-acetyl-L-tryptophan methyl ester and N-acetyl-L-tyrosine ethyl ester catalyzed by alpha-chymotrypsin and delta-chymotrypsin, prepared by fast tryptic activation of nitrated chymotrypsinogen. With both ester substrates Km was doubled for nitrated alpha-chymotrypsin. Nitrated alpha-chymotrypsin, nitrated delta-chymotrypsin and delta-chymotrypsin could all bind N-acetyl-L-tryptophan methyl ester at alkaline pH, in contrast to alpha-chymotrypsin. The dissociation constant, Kd, of the complex of alpha-chymotrypsin and basic pancreatic trypsin inhibitor was lowered ten-fold relative to the constant obtained with unmodified alpha-chymotrypsin. The nitrated delta-chymotrypsin and delta-chymotrypsin showed identical Kd values. The nitrated alpha-chymotrypsin is inactivated faster at pH 8.0 and 8.5 than alpha-chymotrypsin and apparently by a different mechanism.     

Production of Higher-quality Plastein from a Crude Single-cell Protein

From defatted n-paraffin-assimilating yeast cells, a crude protein was obtained by alkaliextraction followed by acid-precipitation. Then the protein was treated with ether until extractable substances were removed exhaustively at this stage. However, at the next stage where the ether-treated protein had been partially hydrolyzed with pepsin, when the hydrolysate was retreated with ether, it was found that ether-extractable substances totalling 270 mg/100 g were obtainable additionally. Chromatographic investigations demonstrated that the substances included significant amounts of aliphatic and aromatic hydrocarbons, some indoles, and a ubiquinone (n = 8).

From the protein hydrolysate (substrate) after the above ether-treatment, a plastein was synthesized with Bioprase under the specific conditions. The plastein was obtained as a precipitate when the whole reaction mixture was treated with aqueous ethanol or acetone. The quantity and quality (nitrogen content) of the plastein depended on the ethanol or acetone concentration. Roughly speaking, the higher the concentration, the more the plastein quantity. The converse relation held for the quality; a plastein precipitated by treatment solely with water showed a higher quality than any other case.     

Production of glucose syrups in highly concentrated systems

We have investigated the hydrolysis of maltodextrins in a high concentration (up to 70%), by means of enzymatic and acid catalysis. The study revealed that the equilibrium compositions of the catalyzed reactions were kinetically determined by the selectivity of the catalyst, the substrate concentration and the reaction time. A model comprising a set of two kinetic equations was used to describe the hydrolysis and condensation reactions of glucoamylase-catalyzed reactions, even to highly concentrated systems. Increased substrate concentration resulted in the formation of more condensation products. The enzyme inhibition was low and was found to be independent of the substrate concentration.     

Mass-transfer and kinetics of microencapsulated alpha-chymotrypsin action]

A theoretical model for transient and steady-state kinetics of microencapsulated enzymes action has been developed. The model is meant to overcome the diffusional limitations, caused by a microcapsulated membrane. The effects of various parameters (enzymatic reaction rate constants, enzyme concentration in microcapsules, membrane permeability, substrate concentration, and bulk pH values) on the overall apparent reaction rate have been analyzed using esters hydrolysis catalyzed by alpha-chymotrypsin encapsulated into polycarbonate membranes as an example.     

A kinetic characterization of the glycosyltransferase activity of Eschericia coli PBP1b and development of a continuous fluorescence assay

The bacterial cell wall is a polymer consisting of alternating N-acetylglucosamine (GlcNAc) and N-acetylmuramic acid (MurNAc) units, cross-linked via peptides appended to MurNAc. The final steps in the formation of cell wall, also referred to as murein, are catalyzed by high-molecular-weight, class A penicillin-binding proteins (PBPs). These bifunctional enzymes catalyze both glycosyltransfer, to form the carbohydrate backbone of murein, and transpeptidation, to form the interstrand peptide linkages. Using PBP1b from Eschericia coli, an in vitro kinetic characterization of the glycosyltransfer reaction was carried out. Initial studies with unlabeled substrate (Lipid II) revealed that activity is strongly influenced by DMSO, as well as metal and detergent. In addition, a continuous fluoresence assay was developed and used to determine the effect of pH on the reaction. A single basic residue was titrated, with a pK(a) of 7.0. Taken together, these data suggest a mechanism for PBP1b where the glycosyltransfer reaction is catalyzed by the concerted effect of an active site base to deprotonate the glycosyl acceptor and a divalent metal to assist departure of the leaving group of the glycosyl donor.     

Stabilization of alpha-chymotrypsin by ionic liquids in transesterification reactions.

Five different ionic liquids, based on dialkylimidazolium and quaternary ammonium cations associated with perfluorinated and bis (trifluoromethyl) sulfonyl amide anions, were used as reaction media to synthesize N-acetyl-L-tyrosine propyl ester by transesterification with alpha-chymotrypsin at 2% (v/v) water content at 50 degrees C. The synthetic activity was reduced by the increase in alkyl chains length of cations and by increases in anion size, which was related to the decrease in polarity. Incubation of the enzyme (with and without substrate) in ionic liquids exhibited first-order deactivation kinetics at 50 degrees C, allowing determination of deactivation rate constants and half-life times (1-3 h). Ionic liquids showed a clear relative stabilization effect on the enzyme, which was improved by increased chain length of the alkyl substituents on the imidazolium ring cations and the anion size. This effect was 10-times enhanced by the presence of substrate. For example, 1-butyl-3-methylimidazolium hexafluorophosphate increased the alpha-chymotrypsin half-life by 200 times in the presence of substrate with respect to the 1-propanol medium. These results show that ionic liquids are excellent enzyme-stabilizing agents and reaction media for clean biocatalysis in non-conventional conditions.     

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.     

The role of hydration in enzyme activity and stability: 1. Water adsorption by alcohol dehydrogenase in a continuous gas phase reactor

The enzymatic synthesis of the tripeptide derivative Z-Gly-Trp-Met-OEt is reported. This tripeptide is a fragment of the cholecystokinin C-terminal octapeptide CCK-8. Studies on the alpha-chymotrypsin catalyzed coupling reaction between Z-Gly-Trp-R(1) and Met-R(2) have focused on low water content media, using deposited enzyme on inert supports such as Celite and polyamide. The effect of additives (polar organic solvents), the acyl-donor ester structure, the C-alpha protecting group of the nucleophile, enzyme loading, and substrate concentration were tested. The best reaction medium found was acetonitrile containing buffer (0.5%, v/v) and triethylamine (0.5%, v/v) using the enzyme deposited on Celite as catalyst (8 mg of alpha-chymotrypsin/g of Celite). A reaction yield of 81% was obtained with Z-Gly-Trp-OCam as acyl donor, at an initial concentration of 80 mM. The tripeptide synthesis was scaled up to the production of 2 g of pure tripeptide with an overall yield of 71%, including reaction and purification steps. (c) 1996 John Wiley & Sons, Inc.     

Reaction mechanism, specificity and pH-dependence of peptide synthesis catalyzed by the metalloproteinase thermolysin

The initial rates of peptide bond formation catalyzed by the metalloproteinase thermolysin were determined. The dependence of the formation rates on the concentration of the carboxyl donor and the acceptor can be explained by a rapid-equilibrium random bireactant mechanism, in which the binding of one substrate has a positive influence on the binding of the other (synergism). The specificity of the enzyme for the donor and acceptor in the condensation reaction was further investigated by determining the apparent kinetic parameters kcat and Km for various substrates. The pH-dependence of the initial rates of synthesis was found to be identical to the pH-dependence of the hydrolytic action of the enzyme. The rates are also shown to be independent of the pKa of the amino group of the acceptor, indicating that deprotonation of the attacking nucleophile in the synthetic reaction is not rate-limiting.     

New fluorogenic substrate for esterase activity of alpha-chymotrypsin and related enzymes

A new fluorogenic substrate, benzyloxycarbonyl-L-phenylalanine 4-methylcoumaryl-7-ester, has been developed for determination of the esterase activity of alpha-chymotrypsin and related enzymes. Synthesis of the substrate was achieved simply by the carbodiimide condensation of benzyloxycarbonyl-L-phenylalanine and 7-hydroxy-4-methylcoumarin in a 86% yield. The esterase activity was measured by increase of the fluorescence intensity at excitation and emission wavelengths of 325 and 465 nm, respectively. An initial rate of hydrolysis was linear over a 100-fold range of the enzyme concentration. As little as 2 ng of alpha-chymotrypsin could be detected in the standard assay. A typical enzyme assay, stability of the substrate, kinetic parameters, and specific activity have been reported.     

Purification and properties of gamma-glutamyltranspeptidase from Proteus mirabilis.

gamma-Glutamyltranspeptidase was purified ca. 15,200-fold from cell-free extracts of Proteus mirabilis to electrophoretic homogeneity and then crystallized. The enzyme has an estimated molecular weight of 80,000 and consists of two different subunits with molecular weights of ca. 47,000 and 28,000. The purified enzyme catalyzed hydrolysis and transpeptidation of various gamma-glutamyl compounds, including the oxidized and reduced forms of glutathione, gamma-glutamyl compounds of L-phenylalanine, L-tyrosine, L-histidine, L-alpha-aminobutyrate, L-leucine, and p-nitroaniline. Glycylglycine, L-phenylalanine, L-methionine, L-histidine, L-tryptophan, and L-isoleucine were good acceptors of the gamma-glutamyl moiety in the transpeptidation reaction. Km values for gamma-glutamyl compounds were on the order of 10(-4) to 10(-5) M, and those for acceptor peptides and amino acids were on the order of 10(-2) to 10(-3) M. The enzyme was inhibited by L-serine plus borate and 6-diazo-5-oxo-L-norleucine, which are inhibitors of gamma-glutamyltranspeptidases isolated from mammals. Various amino acids alone were found to inhibit the transpeptidation competitively with a gamma-glutamyl donor. Kinetic analysis suggested that the reaction sequence of substrate binding and product release proceeds according to a ping pong bi bi mechanism.     

Probes for Catalytic Action of α-Chymotrypsin in Plastein Synthesis

A plastein was synthesized with α-chymotrypsin from a dialyzable fraction of a peptic hydrolysate of soybean protein.

The plastein was obtainable also by use of an insoluble preparation of α-chymotrypsin. This may rule out the possibility that the plastein is a product resulting from some chemical peptide-protein (enzyme) aggregation.

No appreciable amount of the plastein was produced when chymotrypsinogen was used instead of α-chymotrypsin.

The plastein synthetic, as well as the protein hydrolytic, activity of α-chymotrypsin was inhibited more or less by a hydrophobic inhibitor (n-hexane), a competitive inhibitor (benzolyl-d,l-phenylalanine), and divalent cations (Zn²⁺, Hg²⁺ and Cu²⁺); the degree of inhibition in each case was approximately similar against both the synthetic and the hydrolytic activities.

Either diisopropylphosphorylation of the β-O of Ser-195 or methylation of the 3-N of His-57 imidazole of α-chymotrypsin repressed the synthetic, as well as the hydrolytic, activity.

Based on these results a possible mechanism was discussed of the plastein synthesis by α-chymotrypsin, especially in relevance to its acylation and deacylation.     

Divergent effects of compounds on the hydrolysis and transpeptidation reactions of γ-glutamyl transpeptidase

A novel class of inhibitors of the enzyme γ-glutamyl transpeptidase (GGT) were evaluated. The analog OU749 was shown previously to be an uncompetitive inhibitor of the GGT transpeptidation reaction. The data in this study show that it is an equally potent uncompetitive inhibitor of the hydrolysis reaction, the primary reaction catalyzed by GGT in vivo. A series of structural analogs of OU749 were evaluated. For many of the analogs, the potency of the inhibition differed between the hydrolysis and transpeptidation reactions, providing insight into the malleability of the active site of the enzyme. Analogs with electron withdrawing groups on the benzosulfonamide ring, accelerated the hydrolysis reaction, but inhibited the transpeptidation reaction by competing with a dipeptide acceptor. Several of the OU749 analogs inhibited the transpeptidation reaction by slow onset kinetics, similar to acivicin. Further development of inhibitors of the GGT hydrolysis reaction is necessary to provide new therapeutic compounds.     

Large acceleration of alpha-chymotrypsin-catalyzed dipeptide formation by 18-crown-6 in organic solvents

The effects of 18-crown-6 on the synthesis of peptides catalyzed by alpha-chymotrypsin are reported. Lyophilization of the enzyme in the presence of 50 equivalents of 18-crown-6 results in a 425-fold enhanced activity when the reaction between the 2-chloroethylester of N-acetyl-L-phenylalanine and L-phenylalaninamide is carried out in acetonitrile. Addition of crown ether renders the dipeptide synthesis in nonaqueous solvents catalyzed by alpha-chymotrypsin possible on a preparative scale. The acceleration is observed in different solvents and for various peptide precursors. Copyright 1998 John Wiley & Sons, Inc.     

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.     

Salt Effects on Plastein Formation by α-Chymotrypsin

The plastein formation by α-chymotrypsin from an ovalbumin hydrolysate was affected in an order of valency of salts when the concentration of each salt was 1 m. Monovalent cations were rather effective at this concentration and enhanced the plastein yield by 10%. In the presence of NaCl, the plastein formation showed two distinct maximal rates at its concentrations of 0.1 m and 0.8 m. The first maximum was considered to be resulted from an increase in enzyme activity, since chymotryptic hydrolysis of both N-acetyl-l-tyrosine ethyl ester and benzyloxycarbonyl-l-phenylalanine p-nitrophenyl ester was activated at an NaCl concentration of 0.1 ~ 0.2 m. The second maximum was ascribed to the salting-out of the product due to the higher concentration of NaCl. A salt-tolerant protease was also used to confirm the above conclusions. It was observed that this enzyme was much effective in producing a plastein at a high NaCl concentration. This may be due to the fact that both the enzyme activation effect and the product salting-out effect participate co-operatively.