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Probing the Specificity Determinants of Amino Acid Recognition by Arginase

机译：探索精氨酸酶识别氨基酸的特异性决定因素

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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 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.
机译：精氨酸酶是一种双核锰金属酶，可作为治疗哮喘，勃起功能障碍和动脉粥样硬化的治疗靶标。为了更好地理解抑制剂亲和力的分子基础，我们采用了定点诱变，酶动力学和X射线晶体学技术来探测氨基酸部分（即α-氨基和α-羧酸酯基）的分子识别）的底物l-精氨酸和抑制剂在精氨酸酶I的活性位点中。具体而言，我们关注（1）底物α-羧酸盐和T135之间的水介导氢键，（2）底物α之间的直接氢键-羧酸盐和N130，以及（3）底物α-氨基和D183之间的直接电荷氢键。 T135，N130和D183的氨基酸取代通常会损害底物亲和力，如增加的KM值所反映，但对催化功能的影响则不太明显，如kcat的最小变化所反映。与底物KM值一样，抑制剂Kd值会增加与酶突变体的结合，表明精氨酸酶活性位点中分子间相互作用对氨基酸亲和力的相对贡献是水介导的氢键<直接氢键<直接带电氢键。精氨酸酶与相关的双核锰金属酶胍基酶和蛋白原水解min胺水解酶的结构比较表明，精氨酸酶折叠中底物识别的进化是由于活性位点口侧的特异性环（尤其是环4和5）中所含残基的突变而发生的，从而可以容纳各种胍基底物进行催化。

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