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

Author(s)

Edward L. D'Antonio, University of South Carolina - BeaufortFollow
Yang Hai, University of Pennsylvania
David W. Christianson, University of PennsylvaniaFollow

Document Type

Article

Subject Area(s)

Biochemistry

Abstract

Various binuclear metal ion clusters and complexes have been reconstituted in crystalline human arginase I by removing the Mn²⁺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 Mn²⁺_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 Mn²⁺₂, Co²⁺₂, Ni²⁺₂, and Zn²⁺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⁺ ≈ Co²⁺ Zn²⁺. Therefore, Mn²⁺ is required for optimal catalysis by human arginase I.

Publication Info

Published in Biochemistry, Volume 51, Issue 42, 2012, pages 8399-8409.

© Biochemistry 2012, American Chemical Society.

DOI: http://www.doi.org/10.1021/bi301145n