RAT-LIVER ARGINASE - KINETIC MECHANISM, ALTERNATE SUBSTRATES, AND INHIBITORS

The activity of guanidino compounds as alternate substrates for rat liver arginase is critically dependent on the length of the amino acid side chain and the substituents about C-alpha. In addition to L-arginine, the enzyme catalyzes the hydrolysis of L-argininamide, L-canavanine, L-homoarginine, L-argininic acid, and agmatine. The k(cat) values for these substrates are 15- to 5000-fold slower than the k(cat) for L-arginine. Guanidobutyrate, D-arginine, and N-G-methyl-L-arginine are not substrates. Competitive inhibition by the products L-ornithine and urea indicates a rapid-equilibrium random mechanism for the enzyme. Despite the requirement for added divalent cations in the activation of the enzyme, metal chelators such as EDTA and citrate do not inhibit the enzyme. These results suggest that the metal site is not readily accessible to solvent. Multiple inhibition experiments with the noncompetitive inhibitor berate demonstrate that berate and urea bind in a mutually exclusive manner, while L-ornithine and berate can bind simultaneously to the enzyme. Berate inhibition is proposed to arise from chelation of Mn(II) in the binuclear Mn(II) center, thus displacing a metal-bound water molecule that is responsible for nucleophilic attack on the guanidium carbon. (C) 1994 Academic Press, Inc.