Structural studies on ADP activation of mammalian glutamate dehydrogenase and the evolution of regulation
Structural studies on ADP activation of mammalian glutamate dehydrogenase and the evolution of regulation
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DOI：
10.1021/bi0206917
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发表时间：
2003-04-01
期刊：
影响因子：
2.9
通讯作者：
Smith, TJ
中科院分区：
文献类型：
--
作者：
Banerjee, S;Schmidt, T;Smith, TJ
谷氨酸脱氢酶广泛存在于所有生物体中，催化L-谷氨酸可逆氧化脱氨为2-羟基戊二酸。与细菌的GDH不同，哺乳动物的GDH在辅酶、ADP激活和GTP抑制方面表现出负的协同作用。本文介绍了载脂蛋白-牛-谷氨酸脱氢酶、牛-谷氨酸脱氢酶与ADP的络合物，以及对ADP激活不敏感的人-谷氨酸脱氢酶R463A突变体(HuGDH)的结构。在没有活性中心配体的情况下，催化裂解处于开放构象，六聚体在晶胞中形成长聚合物，相互作用比在流产的复杂晶体中发现的更多。这与ADP促进溶液中聚集的事实是一致的。ADP被证明与第二个抑制性NADH结合，但仍可引起激活。结合的ADP的β-磷酸与枢轴螺旋上的R459(huGDH中的R463)相互作用。抗ADP的人GDHR463A突变体除了枢轴螺旋上的侧链被截断外，其他结构与天然GDH一模一样。综上所述，这些结果强烈表明ADP通过促进催化裂隙的打开而激活。从不同来源的GDH的比对来看，很可能是在嘌呤调节位点形成之前，天线在原生生物中进化。这表明，天线本身有一定的选择性优势，动物通过增加变构调节为GDH进化了新的功能。
Glutamate dehydrogenase (GDH) is found in all organisms and catalyzes the reversible oxidative deamination Of L-glutamate to 2-oxoglutarate. Unlike GDH from bacteria, mammalian GDH exhibits negative cooperativity with respect to coenzyme, activation by ADP, and inhibition by GTP. Presented here are the structures of apo bovine GDH, bovine GDH complexed with ADP, and the R463A mutant form of human GDH (huGDH) that is insensitive to ADP activation. In the absence of active site ligands, the catalytic cleft is in the open conformation, and the hexamers form long polymers in the crystal cell with more interactions than found in the abortive complex crystals. This is consistent with the fact that ADP promotes aggregation in solution. ADP is shown to bind to the second, inhibitory, NADH site yet causes activation. The beta-phosphates of the bound ADP interact with R459 (R463 in huGDH) on the pivot helix. The structure of the ADP-resistant, R463A mutant of human GDH is identical to native GDH with the exception of the truncated side chain on the pivot helix. Together, these results strongly suggest that ADP activates by facilitating the opening of the catalytic cleft. From alignment of GDH from various sources, it is likely that the antenna evolved in the protista prior to the formation of purine regulatory sites. This suggests that there was some selective advantage of the antenna itself and that animals evolved new functions for GDH through the addition of allosteric regulation.