Krebs H, Rudolph R, Jaenicke R
Eur J Biochem. 1979 Oct 15;100(2):359-64. doi: 10.1111/j.1432-1033.1979.tb04178.x.
Kinetic analysis of the reactivation in vitro of glyceraldehyde-3-phosphate dehydrogenase from yeast in the presence of NAD+ suggested that transconformation reactions of inactive monomers and their subsequent association to native tetramers are responsible for the sigmoidal relaxations [R. Rudolph et al. (1977) Eur. J. Biochem. 81, 563-570]. Comparison with the reactivation behaviour in the absence of coenzyme was not feasible at this stage due to the instability of the apoenzyme. In the present study, solvent conditions were established which allowed both apoenzyme and holoenzyme to exhibit high stability. The apoenzyme is stable in phosphate buffer; but if excess NAD+ and phosphate are present (both of which stabilize the enzyme if applied separately), destabilization occurs. Protection of functional groups against oxidation by addition of a reducing agent and by degassing and preventing contact with air, increase the stability. Only partial stabilization can be achieved in the presence of NADH. Comparing the kinetics of reactivation in the presence and absence of coenzymes shows that both oxidized and reduced coenzyme enhance the rate of reactivation significantly, and to the same extent. The kinetic effect of coenzyme binding to the refolding polypeptide chain is discussed in terms of the stabilization of intermediates or end products of reconstitution on the one hand, and acceleration of folding and association reactions, on the other.
对酵母中甘油醛-3-磷酸脱氢酶在NAD⁺存在下的体外再活化进行动力学分析表明，无活性单体的构象转变反应及其随后聚合成天然四聚体是导致S形弛豫的原因[R. 鲁道夫等人（1977年），《欧洲生物化学杂志》81卷，563 - 570页]。由于脱辅酶的不稳定性，在这个阶段无法将其与无辅酶时的再活化行为进行比较。在本研究中，建立了溶剂条件，使脱辅酶和全酶都能表现出高稳定性。脱辅酶在磷酸盐缓冲液中稳定；但如果存在过量的NAD⁺和磷酸盐（单独使用时两者都能使酶稳定），则会发生失稳。通过添加还原剂、除气并防止与空气接触来保护官能团免受氧化，可提高稳定性。在NADH存在下只能实现部分稳定。比较有辅酶和无辅酶时的再活化动力学表明，氧化型和还原型辅酶都能显著提高再活化速率，且程度相同。从一方面稳定重构的中间体或终产物，另一方面加速折叠和缔合反应的角度讨论了辅酶与重折叠多肽链结合的动力学效应。
