Abstract

Full activation of human liver arginase (EC 3.5.3.1), by incubation with 5 mM Mn(2+) for 10 min at 60 degrees C, resulted in increased V(max) and a higher sensitivity of the enzyme to borate inhibition, with no change in the K(m) for arginine. Borate behaved as an S-hyperbolic I-hyperbolic non-competitive inhibitor and had no effect on the interaction of the enzyme with the competitive inhibitors L-ornithine (K(i) = 2 +/- 0.5 mM), L-lysine (K(i) = 2.5 +/- 0.4 mM), and guanidinium chloride (K(i) = 100 +/- 10 mM). The pH dependence of the inhibition was consistent with tetrahedral B(OH)(4)(-) being the inhibitor, rather than trigonal B(OH)(3). We suggest that arginase activity is associated with a tightly bound Mn(2+) whose catalytic action may be stimulated by addition of a more loosely bound Mn(2+), to generate a fully activated enzyme form. The Mn(2+) dependence and partial character of borate inhibition are explained by assuming that borate binds in close proximity to the loosely bound Mn(2+) and interferes with its stimulatory action. Although borate protects against inactivation of the enzyme by diethyl pyrocarbonate (DEPC), the DEPC-sensitive residue is not considered as a ligand for borate binding, since chemically modified species, which retain about 10% of enzymatic activity, were also sensitive to the inhibitor. (C)1999 Elsevier Science Inc. All rights reserved.