KINETIC MECHANISM OF GLUTAMATE-DEHYDROGENASE
KINETIC MECHANISM OF GLUTAMATE-DEHYDROGENASE
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DOI：
10.1021/bi00552a007
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发表时间：
1980-01-01
期刊：
影响因子：
2.9
通讯作者：
CLELAND, WW
中科院分区：
文献类型：
--
作者：
RIFE, JE;CLELAND, WW
草酰甘氨酸的初始速度模式和死端抑制研究表明，除了NADPH，酮酸和氨发生强制性的顺序。对于单羧酸酮酸，酮酸-氨的初速度模式是平衡有序的，因为koff远大于Vmax。α-在不存在NADP+的情况下，酮戊二酸产生对NADPH或氨无竞争性的底物抑制，但在高NADP+的情况下，酮戊二酸对NADPH无竞争性，对氨无竞争性。抑制[牛肝酶]在斜率和截距上是部分的，表明酮戊二酸减缓但不阻止NADP+释放，并形成弱但动力学活性的E-酮戊二酸复合物。因此，NADPH和酮酸的表观有序组合是由结合中的强协同作用引起的。氘同位素效应，以及酮戊二酸的底物抑制模式，表明除了氨基酸和NADP+是随机的，与NADP+被释放的三元复合物更迅速地比谷氨酸或正缬氨酸。对于正缬氨酸，氢化物转移是主要的限速步骤，而对于谷氨酸，氢化物转移之前的步骤比氢化物转移慢。平衡~（18）O同位素效应为1.031 ±-。0.006（相对于水，18 O在酮戊二酸中富集），但未观察到动力学18 O同位素效应。
Initial velocity patterns and dead-end inhibition studies with oxalylglycine suggest that addition of NADPH, keto acid and ammonia occurs with obligatory order. For monocarboxylic keto acids, the keto acid-ammonia initial velocity pattern is equilibrium ordered because koff is much greater than Vmax. .alpha.-Ketoglutarate gives substrate inhibition that is uncompetitive vs. either NADPH or ammonia in the absence of NADP+, but with high NADP+ is noncompetitive vs. NADPH and uncompetitive vs. ammonia. The inhibition [of bovine liver enzyme] is partial on slopes and intercepts, showing that ketoglutarate slows down but does not prevent NADP+ release and forms a weak but kinetically competent E-ketoglutarate complex. The apparent ordered combination of NADPH and keto acid thus results from strong synergism in binding. Deuterium isotope effects, together with the substrate inhibition patterns for ketoglutarate, show that addition of amino acid and NADP+ is random, with NADP+ being released from the ternary complex more rapidly than either glutamate or norvaline. With norvaline, hydride transfer is a major rate-limiting step, while with glutamate a step preceding hydride transfer is slower than hydride transfer. The equilibrium 18O siotope effect is 1.031 .+-. 0.006 (18O enriching in ketoglutarate relative to water), but no kinetic 18O isotope effect was seen.