Some kinetic properties of γ-glutamyltransferase from rabbi liver

Abstract

γ-Glutamyltransferase ((5-glutamyl)-peptide: amino-acid 5-glutamyltransferase, EC 2.3.2.2) of rabbit liver (detergent form) was purified 1100-fold in order to study its kinetic properties. Kinetic studies were conducted from pH 6.0 to 12.0 in the absence and presence of the acceptor substrate glycylglycine using γ-glutamyl-3-carboxy-4-nitroanilide as the donor. The existence of more than one binding site for both donor and acceptor is postulated on kinetic evidence such as donor substrate activation, donor substrate inhibition and acceptor substrate activation. Homotropic interaction is also observed, in the form of negative cooperativity, in donor substrate binding, in the absence of acceptor at pH less than 9.0 and positive cooperativity ( n=2), in the absence or presence of acceptor at pH greater than 9.0. Hydrolase reaction reaches a maximum of activity at pH 10 (p K 8.6). Transferase activity under conditions of maximal velocity is maximal at pH 9.0 (p K 7.1). The ratio of transferase activity/hydrolase activity is maximal at pH 7.0–7.5. At low donor substrate concentrations, maximal activity is attained at pH 7.5. Autotransfer is not thought to play a significant role in the conditions used for the study. Studies done with rat kidney brush border membrane at pH 6 and 7.5 corroborate the findings with the purified enzyme.