Cardon J W, Boyer P D
J Biol Chem. 1982 Jul 10;257(13):7615-22.
The evidence for equivalent catalytic sites in tetrameric glyceraldehyde-3-phosphate dehydrogenase from rabbit muscle has been re-examined and found to be insufficient to exclude alternating or reciprocating sites models. Using a column centrifugation technique, lower limits have been set on the rates of binding and release of coenzyme, and on the ratio of the affinities of NAD+ and NADH. The binding to acyl enzyme has also been examined. The tightly bound NAD+ has been found to be reduced preferentially and kinetically competently when glyceraldehyde 3-phosphate is added, demonstrating the nonequivalence of the sites in the transient reduction of NAD+. The rate of release of the NADH formed rapidly from tightly bound NAD+ was monitored directly by using lactate dehydrogenase and pyruvate to regenerate NAD+. This rate was sufficiently rapid for the NADH formed from tightly bound NAD+ to be a catalytic intermediate. Although these and other results are consistent with a simple alternating sites model, additional approaches appear necessary to find if subunit catalytic cooperativity occurs with this enzyme.
兔肌四聚体甘油醛 - 3 - 磷酸脱氢酶中催化位点等效性的证据已被重新审视，发现不足以排除交替或往复位点模型。使用柱离心技术，已确定辅酶结合和释放速率以及NAD⁺和NADH亲和力之比的下限。还研究了与酰基酶的结合。已发现紧密结合的NAD⁺在添加甘油醛3 - 磷酸时优先且在动力学上有效地被还原，这表明在NAD⁺的瞬时还原中位点不等效。通过使用乳酸脱氢酶和丙酮酸再生NAD⁺，直接监测了由紧密结合的NAD⁺快速形成的NADH的释放速率。该速率足够快，使得由紧密结合的NAD⁺形成的NADH成为催化中间体。尽管这些以及其他结果与简单的交替位点模型一致，但似乎需要额外的方法来确定该酶是否存在亚基催化协同作用。
