Structure and Function of Non-Native Metal Clusters in Human Arginase I

Various binuclear metal ion clusters and complexes have been reconstituted in crystalline human arginase I by removing the Mn-2(2+) cluster of the wild type enzyme with metal chelators and subsequently soaking the crystalline apoenzyme in buffer solutions containing NiCl2 or ZnCl2. X-ray crystal structures of these metal ion variants are correlated with catalytic activity measurements that reveal differences resulting from metal ion substitution. Additionally, treatment of crystalline Mn-2(2+)-human arginase I with Zn2+ reveals for the first time the structural basis for inhibition by Zn2+, which forms a carboxylate-histidine-Zn2+ triad with H141 and E277. The imidazole side chain of H141 is known to be hyper-reactive, and its chemical modification or mutagenesis is known to similarly compromise catalysis. The reactive substrate analogue 2(S)-amino-6-boronohexanoic acid (ABH) binds as a tetrahedral boronate anion to Mn-2(2+), Co-2(2+), Ni-2(2+), and Zn-2(2+) clusters in human arginase I, and it can be stabilized by a third inhibitory Zn2+ ion coordinated by H141. Because ABH binds as an analogue of the tetrahedral intermediate and its flanking transition states in catalysis, this implies that the various metallo-substituted enzymes are capable of some level of catalysis with an actual substrate. Accordingly, we establish the following trend for turnover number (k(cat)) and catalytic efficiency (k(cat)/K-M): Mn2+ > Ni2+ approximate to Co2+ >> Zn2+. Therefore, Mn2+ is required for optimal catalysis by human arginase I.