人谷氨酸脱氢酶 (GDH) 变构调控突变的表达、纯化和表征,Biochemical Journal

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人谷氨酸脱氢酶 (GDH) 变构调控突变的表达、纯化和表征
Biochemical Journal ( IF 4.4 ) Pub Date : 2002-03-22 , DOI: 10.1042/bj3630081
Jie FANG ₁ , Betty Y. L. HSU ₁ , Courtney M. MacMULLEN ₁ , Mortimer PONCZ ₂ , Thomas J. SMITH ₃ , Charles A. STANLEY ₁

Affiliation

谷氨酸脱氢酶 (GDH) 催化线粒体基质中 l-谷氨酸到 2-氧代戊二酸的可逆氧化脱氨。在哺乳动物中，这种酶受到变构效应器的高度调节。主要的变构激活剂和抑制剂分别是 ADP 和 GTP；亮氨酸的变构激活可能在氨基酸刺激的胰岛素分泌中起重要作用。通过发现与 GDH 显性突变相关的异常高胰岛素血症/高氨血症综合征的儿童，该调节的生理意义已被突出显示，这些突变导致 GTP 抑制作用的丧失。为了确定这些突变对人类 GDH 同型六聚体功能的影响，我们研究了其中两种调节突变（H454Y、影响假定的 GTP 结合位点和影响天线区域的 S448P）和旨在改变假定的 ADP 结合位点的突变（R463A）。与野生型人 GDH (ED50, 42nM) 或从杂合子患者细胞中分离的 GDH (ED50 ，分别为 290 和 280nM）。对 ADP 或亮氨酸刺激的敏感性不受这些突变的影响，证实它们特异性干扰抑制性 GTP 结合位点。相反，R463A 突变完全消除了人 GDH 的 ADP 活化，但对 GTP 抑制或亮氨酸活化几乎没有影响。这三个突变对 ATP 调节的影响表明，该核苷酸通过其三磷酸尾与 GTP 位点的结合来抑制人 GDH，并且在较高浓度下，通过核苷酸与 ADP 位点的结合激活酶。这些数据基于 X 射线晶体学证实了 GTP 和 ADP 变构调节位点在 GDH 上的分配，并提供了对正负变构控制和人类 GDH 亚基间协同作用所涉及的结构机制的深入了解。

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Expression, purification and characterization of human glutamate dehydrogenase (GDH) allosteric regulatory mutations

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, 210μM) and S448P (ED50, 3.1μM) human GDH mutants compared with the wild-type human GDH (ED50, 42nM) or GDH isolated from heterozygous patient cells (ED50, 290 and 280nM, 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.

更新日期：2002-03-22

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