Probing the Specificity Determinants of Amino Acid Recognition by Arginase

Abstract

Arginase is a binuclear manganese metalloenzyme that serves as a therapeutic target for the treatment of asthma, erectile dysfunction, and atherosclerosis. In order to better understand the molecular basis of inhibitor affinity, we have employed site-directed mutagenesis, enzyme kinetics, and X-ray crystallography to probe the molecular recognition of the amino acid moiety (i.e., the α-amino and α-carboxylate groups) of substrate l-arginine and inhibitors in the active site of human arginase I. Specifically, we focus on: (1) a water-mediated hydrogen bond between the substrate α-carboxylate and T135, (2) a direct hydrogen bond between the substrate α-carboxylate and N130, and (3) a direct charged hydrogen bond between the substrate α-amino group and D183. Amino acid substitutions for T135, N130, and D183 generally compromise substrate affinity as reflected by increased KM values, but have less pronounced effects on catalytic function as reflected by minimal variations of kcat. As with substrate KM values, inhibitor Kd values increase for binding to enzyme mutants and suggest that the relative contribution of intermolecular interactions to amino acid affinity in the arginase active site is: water-mediated hydrogen bond < direct hydrogen bond < direct charged hydrogen bond. Structural comparisons of arginase with the related binuclear manganese metalloenzymes agmatinase and proclavaminic acid amidinohydrolase suggest that the evolution of substrate recognition in the arginase fold occurs by mutation of residues contained in specificity loops flanking the mouth of the active site (especially loops 4 and 5), thereby allowing diverse guanidinium substrates to be accommodated for catalysis.

Crystal structures of Pseudomonas aeruginosa guanidinobutyrase and guanidinopropionase, members of the ureohydrolase superfamily

“…4B) (Shishova et al, 2009). These loops 1 and 2 are positioned between b4 and a5 in GbuA, and b5 and a6 in GpuA, respectively.…”

“…Therefore, these enzymes may have evolved from a common ancestral protein and diverged into their particular functions with different substrate specificities (Arakawa et al, 2003;Dowling et al, 2008;Itoh, 2002, 2005;Sekowska et al, 2000). Previous reports showed that substrate recognition is mainly determined by the loops around the active site, which allows diverse guanidine substrates to be accommodated for catalysis (Ahn et al, 2004;Bewley et al, 1999;Cama et al, 2003;Di Costanzo et al, 2005Elkins et al, 2002;Kanyo et al, 1996;Shishova et al, 2009). Crystal structures of ureohydrolases are available for rat liver arginase I (Kanyo et al, 1996), human arginase I (Di Costanzo et al, 2005), human arginase II (Cama et al, 2003), Bacillus caldovelox arginase (Bewley et al, 1999), Streptomyces clavuligerus PAH (Elkins et al, 2002), and Deinococcus radiodurans agmatinase (Ahn et al, 2004).…”

Manganese and its Role in Parkinson’s Disease: From Transport to Neuropathology

“…Manganese (Mn) is an essential nutrient important to protein and energy metabolism, bone mineralization, metabolic regulation, and cellular protection from reactive oxygen species. It is a cofactor for enzymes, such as mitochondrial superoxide dismutase (Hearn et al 2003), arginase (Shishova et al 2009), pyruvate carboxylase (Zwingmann et al 2004), and glutamine synthetase (Takeda 2003).…”

Crystallization of an apo form of human arginase: using all the tools in the toolbox simultaneously

“…The first X-ray structure of arginase I was of the rat liver enzyme and revealed the bi-atomic manganese centre which is the hallmark of this enzyme (Kanyo et al, 1996). Previous work has provided high-resolution crystal structures of hArginase I with the tight-binding substrate analogues 6-boronohexanoic acid (ABH) and S-(2-boronoethyl)-l-cysteine (BEC), along with various other molecules (Shishova et al, 2009;Ilies et al, 2010;Di Costanzo et al, 2005Baggio et al, 1997). There has also been a report of an unliganded hArginase I structure (PDB code 2pha): in this instance, the protein was crystallized in the presence of a small molecule which was not seen in the subsequent structure (Di Costanzo et al, 2007).…”