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The kinetics of human serum gamma-glutamyltransferase (EC 2.3.2.2) were investigated, with use of glycylglycine as a gamma-glutamyl acceptor substrate and gamma-glutamyl-4-nitroanilide and its carboxy derivative, gamma-glutamyl-3-carboxy-4-nitroanilide, as donor substrates. The simultaneous occurrence of both gamma-glutamyltransfer and autotransfer was established by descending paper chromatography. Constant-ratio double-reciprocal plots confirm that the enzyme mechanism is nonsequential (ping-pong bi-bi). Inhibition by either donor was not found, and inhibition by glycylglycine was only observed at concentrations above those of clinical interest. Kinetic constants obtained by nonlinear regression analysis of initial velocity data were used to determine reagent substrate concentrations for the assay of this enzyme. An assay with use of 4 mmol of gamma-glutamyl-3-carboxy-4-nitroanilide and 100 mmol of glycylglycine per liter yielded equivalent activities to those by assay with use of 4 mmol of gamma-glutamyl-4-nitroanilide and 40 mmol of glycylglycine per liter. These concentrations of the carboxy donor and glycylglycine are also "cost optimal" and present no procedural problems when used.