Abstract
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.
Highlights
When the substrate was incubated with the enzyme in the presence of an amino acid or peptide acceptor, the corresponding
When L-a-methyl-y-glutamyl-L-a-aminobutyrate was incubated with y-glutamyl transpeptidase and the products were studied by paper chromatography in either Solvent A or B, only L-a-methyl glutamate and L-a-aminobutyrate were detected as the products of the reaction
This indicated that the substitution of a methyl group for a hydrogen on the a-carbon abolished the ability of the amino acid to act as an acceptor
Summary
When the substrate was incubated with the enzyme in the presence of an amino acid or peptide acceptor, the corresponding. When the enzyme was incubated with conventional glutathione or y-glutamyl-p-nitroanilide and an amino acid acceptor, hydrolysis, substrates, such as autotranspeptidation, and transpeptidation to the acceptor occurred concurrently. A scheme of such a mechanism is presented This mechanism predicts the formation of an cu-methyl-y-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 y-glutamyl-p-nitroanilide predict either linear or nonlinear double-reciprocal plots, depending on the prevalence of the hydrolytic, autotranspeptidation, or transpeptidation reactions. It has not yet been established which of these reactions predominates under physiological conditions
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