A SLOW OBLIGATORY PROTON RELEASE STEP PRECEDES HYDRIDE TRANSFER IN THE LIVER GLUTAMATE-DEHYDROGENASE CATALYTIC MECHANISM
A SLOW OBLIGATORY PROTON RELEASE STEP PRECEDES HYDRIDE TRANSFER IN THE LIVER GLUTAMATE-DEHYDROGENASE CATALYTIC MECHANISM
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DOI：
10.1016/0167-4838(92)90233-4
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发表时间：
1992-02-13
期刊：
影响因子：
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通讯作者：
SRINIVASAN, R
中科院分区：
文献类型：
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作者：
FISHER, HF;MANISCALCO, S;SRINIVASAN, R
我们用停流指示剂染料法同时测定了谷氨酸脱氢酶催化的L-谷氨酸氧化脱氨反应的初始瞬时部分的质子释放和产物生成。我们观察到一个质子从酶-NADP-L-谷氨酸复合体中缓慢释放出来。从产物形成信号的明显滞后可以看出，这种质子释放先于氢化物转移步骤。我们证明，质子释放步骤对应于反应序列中的一个必需中间体。我们还发现，作为L-谷氨酸的竞争性抑制剂的化合物能够诱导这种现象。我们证明了这种意想不到的预氢化物转移事件不可能是由于α-氨基质子从底物中释放出来的。
We have used the stopped-flow indicator dye method to measure proton release and product formation simultaneously in the initial transient-state portion of the glutamate dehydrogenase-catalyzed oxidative deamination of L-glutamate. We observe a measurably slow release of a proton from the enzyme-NADP-L-glutamate complex. This proton release precedes the hydride transfer step, as indicated by the distinct lag in the product formation signal. We show that the proton release step corresponds to an obligatory intermediate in the reaction sequence. We also find that compounds which are competitive inhibitors of L-glutamate are capable of inducing this phenomenon. We prove that this unanticipated prehydride transfer event cannot be due to the release of an alpha-amino group proton from the substrate.