Determination of the mechanism and kinetic constants for hog kidney γ-glutamyltransferase

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Abstract
The initial-velocity kinetics of hog kidney .gamma.-glutamyltransferase, (EC 2.3.2.2) were studied. Glutamate .gamma.-(4-nitroanilide) and its 3-carboxy derivative, glutamate .gamma.-(3-carboxy-4-nitroanilide), served as .gamma.-glutamyl donors, and glycylglycine as an acceptor. Reaction products were identified by paper chromatography and amino acid analysis. Inhibited Ping Pong mechanisms and a comprehensive initial-velocity expression were developed which account for the observed simultaneous .gamma.-glutamyl transfer and autotransfer, competitive inhibition by glycylglycine, and non-competitive inhibition by the carboxy donor. The validity of the proposed Ping Pong mechanisms are supported by enzyme-velocity data obtained with constant ratios of acceptor to donor concentrations. Kinetic constants were determined by a non-linear regression analysis. With glutamate .gamma.-(4-nitroanilide) as the donor, Km values for the donor, acceptor and donor-acting-as-acceptor are 1.87, 24.9 and 2.08 mM, respectively. With glutamate .gamma.-(3-carboxy-4-nitroanilide) as the donor, these Km values are 1.63, 16.6 and 12.3 mM. Glycylglycine competitive inhibition constants with the parent donor and its carboxy derivative are 275 and 205 mM, respectively; the non-competitive inhibition constant of the carboxy donor is 34 mM.