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Probing trypsin specificity with basic 3-substituted glutarate diesters and related monoester substrate analogs: Evidence for allosteric activation of trypsin catalysis

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Abstract

A series of dimethyl glutarates bearing basic substituents at C-3, and related monoesters, have been evaluated as substrates of trypsin in order to probe the asymmetric synthetic potential of the enzyme with respect to enantiotopic ester group, and enantiomer, discrimination. While none of the mono- or diesters proved to be a trypsin substrate, several of them accelerated trypsin-catalyzed hydrolysis of the standard reference substrate BAEE, in a manner consistent with an allosteric activation process. The results provide the first examples of allosteric activation of trypsin by modifiers that are sterically precluded from interacting effectively at the acive site.

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The effect of 1- and 2-alkylamines on the tryptic hydrolysis of neutral substrates has been investigated. With acetylglycine p-nitrophenyl ester as substrate, 2-alkylamines have been found to be more efficient activators than the corresponding 1-alkylamines. In contrast, the latter amines are bound to the enzyme with greater affinity than the former. 2-Heptylamine is the most powerful activator of the tryptic hydrolysis of acetylglycine ethyl and p-nitrophenyl esters (activation up to 45-fold under conditions where [S] < Km). The imidazole pK shift to the acidic region has been found to be larger with 2-heptylamine (ΔpK = -0.69) than with ethylamine (ΔpK = -0.31). In the ternary complex formed with enzyme, 2-heptylamine, and acetylglycine p-nitrophenyl ester, the catalytic activity is increased by at least two orders of magnitude and is close to that of specific substrates. All amines studied increase both kcat and Km for the hydrolysis of acetylglycine p-nitrophenyl ester. In the case of acetylglycine ethyl ester, Km is significantly decreased in the presence of 2-heptylamine. Evidence is presented that activation by amines is greater for the deacylation step than for the acylation. The affinity of 2-heptylamine toward enzyme appears to be dependent on the type of enzyme intermediate. The order of decreasing stabilities for amine complexes in this case are Michaelis complex-2-heptylamine > free enzyme-2-heptylamine > acyl-enzyme-2-heptylamine. It is suggested that the active-site conformation is modified during the catalytic processes. The hydrolysis of p-nitrophenyl acetate is only slightly activated by amines, which partially inhibit the hydrolysis of ethyl hippurate. These results are consistent with the existence of an auxiliary binding site for neutral substrates which can interact with a primary site occupied by amines.
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The relationship was investigated between the molecular structure and the antistaphylococcal and antifibrinolytic actions of ω-amino acids and their L-histidine dipeptides, of which δ-aminovaleryl-L-histidine (9) and ε-aminocaproyl-L-histidine (10) are newly synthesized. The antistaphylococcal properties were demonstrated through their protective effects against staphylococcal infections in mice. The antifibrinolytic activities were determined in vitro by measuring prolongation of lysis time of a fibrin clot. The order of antistaphylococcal potencies of these compounds was: (a) glycine (1) < β-alanine (2) < γ-aminobutyric acid (3) < ε-aminocaproic acid (5) < δ-aminovaleric acid (4) and (b) glycyl-L-histidine (6) < β-alanyl-L-histidine (7) < γ-aminobutyryl-L-histidine (8) < δ-aminovaleryl-L-histidine (9) < ε-aminocaproyl-L-histidine (10). Comparing a and b, the protective power of ω-aminoacyl-L-histidines was much higher than that of the corresponding ω-amino acids. The order of antifibrinolytic potencies of ω-amino acids was identical with that of antistaphylococcal action except that 4 < 5 in the former. Practically no antifibrinolytic activity of ω-aminoacyl-L-histidines was observed in vitro under the conditions we employed.
Article
In an attempt to assess the role of the positive charge in trypsin specificity, the hydrolysis constants of neutral substrates were compared with their charged analogs. Replacement of the side chain of arginine by that of citrulline and of lysine by that of heptyline resulted in decreases of 102-106 in k0/K0. Enzyme treated to remove chymotrypsin impurities did not lose activity toward these substrates. Trypsin inactivated with diisopropylphosphofluoridate or 1-chloro-3-tosylamido-7-amino-2-heptanone failed to hydrolyze the neutral substrates. Ternary complex formation involving the enzyme, a positively charged molecule, and a neutral molecule was indicated by the follow-ing data. (1) The rate of hydrolysis of a given substrate was affected in a qualitatively different manner by modifiers of different charge types at equivalent [M]/Ki ratios: phenol was a competitive inhibitor of benzoylcitrulline methyl ester hydrolysis while phenylguanidinium chloride was a noncompetitive inhibitor. (2) A given modifier affected the hydrolysis of different substrate charge types in a qualitatively different manner: phenol was a competitive inhibitor of benzoylcitrulline methyl ester hydrolysis and a noncompetitive inhibitor of benzoylarginine methyl ester hydrolysis. An auxiliary binding site to which neutral molecules preferentially bind is thus implicated.
Article
A series of alkylguanidine hydrochlorides has been prepared, and their inhibitory action against trypsin catalysis has been studied. Inhibition constants obtained have indicated alkylguanidines are stronger inhibitors than the corresponding amines. Methyl- and ethylguanidines increase the rate of the hydrolysis of acetylglycine ethyl ester catalyzed by trypsin seven- and twofold, respectively. However, the extents of the activation are less than that obtainable with ethylamine. The dissociation constant of the methylguanidine-trypsin complex in the activation is identical with the inhibition constant, indicating that binding of the recognition site of enzyme causes the activation. The activation of the acetylglycine ethyl ester hydrolysis by methylguanidine and ethylamine has been studied as a function of temperature and pH. Results have been obtained which indicate that a shift of the pH-activity profile to a lower pH is a factor contributing to the apparent increase in the rate in the activated catalysis. However, the major factor causing the increased rate seems to be an increase in the entropy of activation and not a decrease in the heat of activation, as suggested by a temperature independence of the relative increment of the rate of the acetylglycine ethyl ester hydrolysis in the activation experiment using methylguanidine and ethylamine as activators.
Article
Immobilized trypsin covalently attached to cross-linked dextran was prepared. In the presence of high concentrations of primary and secondary alcohols, the transesterification of L-lysine esters catalyzed by the immobilized trypsin was observed to be predominant. In contrast to this, no transesterified product was detected with tertiary butyl alcohol. These reactions were compared with the acid-catalyzed transesterification reactions and the characteristics of the enzymatic reaction are discussed.
Article
The paper deals with the effect of changes in the concentration of carbonic acid in the medium on the reaction rate catalyzed with enzymes of various spectrum of the action. It is shown that the presence of carbonic acid in the medium reaction increases the rate of reactions catalyzed with lactate dehydrogenase of the rabbit liver soluble fraction, with glucose-6-phosphate dehydrogenase from yeast and trypsin. Under the same conditions the reaction rate catalyzed with glucose-6-phosphate dehydrogenase of the rabbit liver soluble fraction and with ATP-citrate (pro-3S)-lyase is considerably decreased. Changes in the carbonic acid concentrations within the physiological limits are found to have no effect on lactate dehydrogenase from the cattle heart and chymotrypsin.
Article
1. We report the trypsin-catalysed conversion, in high yield, of peptides to peptide hydrazides, t-butyloxycarbonylhydrazides and phenylhydrazides. The substitution is at the alpha-carboxy group. 2. We discuss the relative merits of carrying out the conversion either simultaneously with tryptic cleavage of the parent protein or after such cleavage. 3. We report analogous results with chymotrypsin, elastase and subtilisin. 4. We propose the use of such products in protein semi-synthesis and in the preparation of specific proteinase inhibitors.
Article
This chapter explains that enzyme-catalyzed chemical modification of proteins is an important mechanism for the regulation of their biological activity. It discusses that the chemical modification reactions, phosphorylation and dephosphorylation, are coupled to various physiological stimuli and that the modification reactions have an important consequence on glycogen metabolism. This type of control is now known to be quite common for other enzymes and proteins, because these enzyme-catalyzed chemical modification reactions are important in regulatory phenomena. The chapter focuses on the use of alternative substrates. It discusses the several important ways that alternative substrates can be used. It is important to establish whether the alternative substrate chosen is a useful model for the study of the enzyme-catalyzed modification reaction. Finally, it concludes that the use of alternative substrates in conjunction with natural substrates in the study of enzymic covalent modification reactions has important value. The determination of the site of action of a modifier is an important goal in terms of understanding enzyme regulation. The next phase and a more complex part is the analysis of the action of these modifiers. Kinetics is just one approach that can be taken to understand the molecular basis of the modifier action.