Peptide mapping identifies hotspot site of modification in human serum albumin by methylglyoxal involved in ligand binding and esterase activity
Peptide mapping identifies hotspot site of modification in human serum albumin by methylglyoxal involved in ligand binding and esterase activity
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DOI：
10.1074/jbc.m410973200
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发表时间：
2005-02-18
影响因子：
4.8
通讯作者：
Thornalley, PJ
中科院分区：
文献类型：
--
作者：
Ahmed, N;Dobler, D;Thornalley, PJ
丙酮醛是一种在生理条件下有效的糖化剂。丙酮醛对人血清白蛋白的体内修饰。由甲基乙二醛修饰所形成的糖基化加合物以及诱导的结构和功能变化尚未完全公开。丙酮醛在生理条件下与人血清白蛋白反应，主要生成氢咪唑酮N-δ-（5-氢-5-甲基-4-咪唑啉-2-基）-鸟氨酸（占总修饰量的92%），少量生成精氨酸嘧啶（N-是-（1-羧乙基）赖氨酸的一个元素）和丙酮醛赖氨酸二聚体。当人血清白蛋白修饰最低限度的甲基乙二醛，胰蛋白酶肽图谱表明，在Arg-410位于药物结合位点II和白蛋白相关酯酶活性的活性位点的修饰热点。在体内糖化的白蛋白中发现了丙酮醛对Arg-410的修饰。其他轻微修饰的位点为：Arg-114、Arg-186、Arg-218和Arg-428。在Arg-410处形成的氢咪唑酮抑制酮洛芬结合和酯酶活性;相应地，在酮洛芬存在下的糖基化抑制Arg-410修饰和酯酶活性的丧失。酯酶活性的pH依赖性表明具有pK（a）= 7.9 +/- 0.1的催化基团，归属于催化碱Tyr-411，其中共轭碱通过与Arg-410的胍基相互作用而稳定。通过甲基乙二醛的修饰使Tyr-411不稳定并将pK增加至8.8 +/- 0.1。分子动力学和建模研究表明，氢咪唑酮的形成导致结构扭曲，从而导致干扰的奎宁定向氢键和静电相互作用的损失。因此，无论是实验性的还是生理性的，关键精氨酸残基的甲基乙二醛修饰预期会破坏蛋白质-配体相互作用并通过氢咪唑酮形成抑制酶活性。
Methylglyoxal is a potent glycating agent under physiological conditions. Human serum albumin is modified by methylglyoxal in vivo. The glycation adducts formed and structural and functional changes induced by methylglyoxal modification have not been fully disclosed. Methylglyoxal reacted with human serum albumin under physiological conditions to form mainly the hydroimidazolone N-delta-(5-hydro-5-methyl-4-imidazolon-2-yl)-ornithine (92% of total modification) with a minor formation of argpyrimidine, N-is an element of-(1-carboxyethyl)lysine, and methylglyoxal lysine dimer. When human serum albumin was modified minimally with methylglyoxal, tryptic peptide mapping indicated a hotspot of modification at Arg-410 located in drug-binding site II and the active site of albumin-associated esterase activity. Modification of Arg-410 by methylglyoxal was found in albumin glycated in vivo. Other sites of minor modification were: Arg-114, Arg-186, Arg-218, and Arg-428. Hydroimidazolone formation at Arg-410 inhibited ketoprofen binding and esterase activity; correspondingly, glycation in the presence of ketoprofen inhibited Arg-410 modification and loss of esterase activity. The pH dependence of esterase activity indicated a catalytic group with pK(a) = 7.9 +/- 0.1, assigned to the catalytic base Tyr-411 with the conjugate base stabilized by interaction with the guanidinium group of Arg-410. Modification by methylglyoxal destabilized Tyr-411 and increased the pK, to 8.8 +/- 0.1. Molecular dynamics and modeling studies indicated that hydroimidazolone formation caused structural distortion leading to disruption of arginine-directed hydrogen bonding and loss of electrostatic interactions. Methylglyoxal modification of critical arginine residues, therefore, whether experimental or physiological, is expected to disrupt protein-ligand interactions and inactivate enzyme activity by hydroimidazolone formation.