Forte-McRobbie C, Pietruszko R
Center of Alcohol Studies, Rutgers University, Piscataway, NJ 08855-0969.
Biochem J. 1989 Aug 1;261(3):935-43. doi: 10.1042/bj2610935.
The kinetic mechanism of homogeneous human glutamic-gamma-semialdehyde dehydrogenase (EC 1.5.1.12) with glutamic gamma-semialdehyde as substrate was determined by initial-velocity, product-inhibition and dead-end-inhibition studies to be compulsory ordered with rapid interconversion of the ternary complexes (Theorell-Chance). Product-inhibition studies with NADH gave a competitive pattern versus varied NAD+ concentrations and a non-competitive pattern versus varied glutamic gamma-semialdehyde concentrations, whereas those with glutamate gave a competitive pattern versus varied glutamic gamma-semialdehyde concentrations and a non-competitive pattern versus varied NAD+ concentrations. The order of substrate binding and release was determined by dead-end-inhibition studies with ADP-ribose and L-proline as the inhibitors and shown to be: NAD+ binds to the enzyme first, followed by glutamic gamma-semialdehyde, with glutamic acid being released before NADH. The Kia and Kib values were 15 +/- 7 microM and 12.5 microM respectively, and the Ka and Kb values were 374 +/- 40 microM and 316 +/- 36 microM respectively; the maximal velocity V was 70 +/- 5 mumol of NADH/min per mg of enzyme. Both NADH and glutamate were product inhibitors, with Ki values of 63 microM and 15,200 microM respectively. NADH release from the enzyme may be the rate-limiting step for the overall reaction.
以谷氨酸γ-半醛为底物，通过初速度、产物抑制和终产物抑制研究确定了人源均相谷氨酸γ-半醛脱氢酶（EC 1.5.1.12）的动力学机制为强制有序，三元复合物可快速相互转化（Theorell-Chance机制）。用NADH进行的产物抑制研究表明，相对于不同浓度的NAD⁺呈竞争性抑制模式，相对于不同浓度的谷氨酸γ-半醛呈非竞争性抑制模式；而用谷氨酸进行的产物抑制研究表明，相对于不同浓度的谷氨酸γ-半醛呈竞争性抑制模式，相对于不同浓度的NAD⁺呈非竞争性抑制模式。通过以ADP-核糖和L-脯氨酸作为抑制剂的终产物抑制研究确定了底物结合和释放的顺序，结果表明：NAD⁺首先与酶结合，随后是谷氨酸γ-半醛，谷氨酸在NADH释放之前被释放。Kia和Kib值分别为15±7 μM和12.5 μM，Ka和Kb值分别为374±40 μM和316±36 μM；最大速度V为每毫克酶每分钟70±5 μmol NADH。NADH和谷氨酸均为产物抑制剂，Ki值分别为63 μM和15200 μM。NADH从酶上的释放可能是整个反应的限速步骤。
