Expression, purification and characterization of human glutamate dehydrogenase (GDH) allosteric regulatory mutations
Expression, purification and characterization of human glutamate dehydrogenase (GDH) allosteric regulatory mutations
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DOI：
10.1042/0264-6021:3630081
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发表时间：
2002-04-01
影响因子：
4.1
通讯作者：
Stanley, CA
中科院分区：
文献类型：
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作者：
Fang, J;Hsu, BYL;Stanley, CA
谷氨酸脱氢酶催化线粒体基质中L-谷氨酸可逆氧化脱氨为2-羟基戊二酸。在哺乳动物中，这种酶受到变构效应器的高度调控。主要的变构激活剂和抑制剂分别是ADP和GTP；亮氨酸对变构的激活可能在氨基酸刺激的胰岛素分泌中起重要作用。这一调节的生理学意义已经通过发现患有不寻常的高胰岛素/高氨血症综合征的儿童而得到强调，这种综合征与导致GTP抑制丧失的GDH显性突变有关。为了确定这些突变对人类GDH同源六聚体功能的影响，我们研究了其中两个调节突变(H454Y，影响推测的GTP结合位点，以及S448P，影响天线区域)和一个突变(R463A)的表达、纯化和特性。纯化的H454Y(ED50,210um)和S448P(ED50，3.1um)突变体对GTP抑制的敏感性明显低于野生型(ED50，42 nM)或从杂合子患者细胞中分离的GDH(ED50,290和280 nM)。对ADP或亮氨酸刺激的敏感性不受这些突变的影响，证实它们特异性地干扰了抑制性GTP结合部位。相反，R463A突变完全消除了人GDH的ADP激活，但对GTP抑制或亮氨酸激活几乎没有影响。这三个突变对ATP调节的影响表明，该核苷酸通过将其三磷酸尾巴与GTP位点结合来抑制人GDH，并在较高浓度下通过与ADP位点结合而激活该酶。这些数据证实了基于X射线结晶学的GDH上GTP和ADP变构调节位点的分配，并为深入了解人类GDH的正负变构控制和亚基间协同作用所涉及的结构机制提供了线索。
Glutamate dehydrogenase (GDH) catalyses the reversible oxidative deamination Of L-glutamate to 2-oxoglutarate in the mitochondrial matrix. In mammals, this enzyme is highly regulated by allosteric effectors. The major allosteric activator and inhibitor are ADP and GTP, respectively; allosteric activation by leucine may play an important role in amino acid-stimulated insulin secretion. The physiological significance of this regulation has been highlighted by the identification of children with an unusual hyperinsulinism/hyperammonaemia syndrome associated with dominant mutations in GDH that cause a loss in GTP inhibition. In order to determine the effects of these mutations on the function of the human GDH homohexamer, we studied the expression, purification and characterization of two of these regulatory mutations (H454Y, which affects the putative GTP-binding site, and S448P, which affects the antenna region) and a mutation designed to alter the putative binding site for ADP (R463A). The sensitivity to GTP inhibition was impaired markedly in the purified H454Y (ED50, 210 muM) and S448P (ED50, 3.1 muM) human GDH mutants compared with the wild-type human GDH (ED50, 42 nM) or GDH isolated from heterozygous patient cells (ED50, 290 and 280 nM, respectively). Sensitivity to ADP or leucine stimulation was unaffected by these mutations, confirming that they interfere specifically with the inhibitory GTP-binding site. Conversely, the R463A mutation completely eliminated ADP activation of human GDH, but had little effect on either GTP inhibition or leucine activation. The effects of these three mutations on ATP regulation indicated that this nucleotide inhibits human GDH through binding of its triphosphate tail to the GTP site and, at higher concentrations, activates the enzyme through binding of the nucleotide to the ADP site. These data confirm the assignment of the GTP and ADP allosteric regulatory sites on GDH based on X-ray crystallography and provide insight into the structural mechanisms involved in positive and negative allosteric control and in inter-subunit co-operativity of human GDH.