Probing the specificity determinants of amino acid recognition by arginase.
Probing the specificity determinants of amino acid recognition by arginase.
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DOI：
10.1021/bi801911v
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发表时间：
2009-01-13
期刊：
影响因子：
2.9
通讯作者：
Christianson, David W.
中科院分区：
文献类型：
--
作者：
Shishova, Ekaterina Y.;Di Costanzo, Luigi;Emig, Francis A.;Ash, David E.;Christianson, David W.
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点。为了更好地了解抑制剂亲和力的分子基础，我们采用定点诱变、酶动力学、x射线晶体学等方法，探索了底物l-精氨酸和抑制剂在人精氨酸酶i活性位点的氨基酸部分（即α-氨基和α-羧酸基）的分子识别。(1)底物α-羧酸酯与T135之间有一个水介导的氢键，(2)底物α-羧酸酯与N130之间有一个直接氢键，(3)底物α-氨基与D183之间有一个直接带电氢键。T135、N130和D183的氨基酸取代通常会损害底物亲和力，这反映在KM值的增加上，但对催化功能的影响不太明显，这反映在kcat的最小变化上。与底物KM值一样，抑制剂Kd值随着与酶突变体的结合而增加，这表明分子间相互作用对精氨酸酶活性位点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agmatinase和proclavaminic amidinohydrolase的结构比较表明，精氨酸酶折叠中底物识别的进化是通过活性位点口侧特异性环（特别是环4和环5）中所含残基的突变发生的，从而允许多种胍底物被容纳催化。
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.
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影响因子：
4.1
作者：
HERZFELD, A;RAPER, SM
通讯作者：
RAPER, SM
影响因子：
15
作者：
Baggio, R;Elbaum, D;Christianson, DW
通讯作者：
Christianson, DW
DOI：
10.1016/s0969-2126(99)80056-2
发表时间：
1999-04-15
期刊：
STRUCTURE WITH FOLDING & DESIGN
影响因子：
--
作者：
Bewley, MC;Jeffrey, PD;Baker, EN
通讯作者：
Baker, EN
影响因子：
15
作者：
Di Costanzo, Luigi;Pique, Michael E.;Christianson, David W.
通讯作者：
Christianson, David W.
DOI：
10.1107/s0907444998003254
发表时间：
1998-09-01
期刊：
ACTA CRYSTALLOGRAPHICA SECTION D-BIOLOGICAL CRYSTALLOGRAPHY
影响因子：
--
作者：
Brunger, AT;Adams, PD;Warren, GL
通讯作者：
Warren, GL