Johnson S L, Tuazon P T
Biochemistry. 1977 Mar 22;16(6):1175-83. doi: 10.1021/bi00625a023.
The rate of the primary acid modification reaction of 1,4-dihydronicotinamide adenine dinucleotide (NADH) and 1,4-dihydro-3-acetylpyridine adenine dinucleotide (APADH) and their analogues has been studied over a wide pH range (pH 1-7) with a variety of general acid catalysts. The rate depends on [H+] at moderate pH and becomes independent of [H+] at low pH. This behavior is attributed to substrate protonation at the carbonyl group (pK of NADH = 0.6). The reaction is general acid catalyzed; large solvent deuterium isotope effects are observed for the general acid and lyonium ion terms. Most buffers cause a linear rate increase with increasing buffer concentration, but certain buffers cause a hyperbolic rate increase. The nonlinear buffer effects are due to complexation of the buffer with the substrate, rather than to a change in rate-limiting step. The rate-limiting step is a proton transfer from the general acid species to the C5 position of the substrate. Anomerization is not a necessary first step in the case of the primary acid modification reaction of beta-NADH, in which beta to alpha anomerization takes place.
在较宽的pH范围（pH 1 - 7）内，使用多种广义酸催化剂研究了1,4 - 二氢烟酰胺腺嘌呤二核苷酸（NADH）和1,4 - 二氢 - 3 - 乙酰吡啶腺嘌呤二核苷酸（APADH）及其类似物的初级酸修饰反应速率。在中等pH值下，反应速率取决于[H⁺]，而在低pH值下则与[H⁺]无关。这种行为归因于羰基处的底物质子化（NADH的pK = 0.6）。该反应是广义酸催化的；对于广义酸和鎓离子项观察到较大的溶剂氘同位素效应。大多数缓冲剂会随着缓冲剂浓度的增加使反应速率呈线性增加，但某些缓冲剂会使反应速率呈双曲线增加。非线性缓冲效应是由于缓冲剂与底物的络合，而不是由于限速步骤的改变。限速步骤是质子从广义酸物种转移到底物的C5位置。在β - NADH的初级酸修饰反应中，β到α的异头物异构化并非必要的第一步，在此反应中会发生β到α的异头物异构化。
