JBC, Vol. 251, Issue 15, 4736-4743, Aug, 1976
Kinetic studies of sheep kidney gamma-glutamyl transpeptidase
A. M. Karkowsky, M. V. Bergamini and M. Orlowski
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.
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