Donoso J, Muñoz F, García Del Vado A, Echevarría G, García Blanco F
Biochem J. 1986 Aug 15;238(1):137-44. doi: 10.1042/bj2380137.
Formation and hydrolysis rate constants as well as equilibrium constants of the Schiff base derived from pyridoxal 5'-phosphate and n-hexylamine were determined between pH 3.5 and 7.5 in ethanol/water mixtures (3:17, v/v, and 49:1, v/v). The results indicate that solvent polarity scarcely alters the values of these constants but that they are dependent on the pH. Spectrophotometric titration of this Schiff base was also carried out. We found that a pKa value of 6.1, attributed in high-polarity media to protonation of the pyridine nitrogen atom, is independent of solvent polarity, whereas the pKa of the monoprotonated form of the imine falls from 12.5 in ethanol/water (3:17) to 11.3 in ethanol/water (49:1). Fitting of the experimental results for the hydrolysis to a theoretical model indicates the existence of a group with a pKa value of 6.1 that is crucial in the variation of kinetic constant of hydrolysis with pH. Studies of the reactivity of the coenzyme (pyridoxal 5'-phosphate) of glycogen phosphorylase b with hydroxylamine show that this reaction only occurs when the pH value of solution is below 6.5 and the hydrolysis of imine bond has started. We propose that the decrease in activity of phosphorylase b when the pH value is less than 6.2 must be caused by the cleavage of enzyme-coenzyme binding and that this may be related with protonation of the pyridine nitrogen atom of pyridoxal 5'-phosphate.
在乙醇/水混合物（体积比3:17和49:1）中，测定了pH值在3.5至7.5之间时，由磷酸吡哆醛和正己胺衍生的席夫碱的形成速率常数、水解速率常数以及平衡常数。结果表明，溶剂极性几乎不会改变这些常数的值，但它们取决于pH值。还对该席夫碱进行了分光光度滴定。我们发现，在高极性介质中，归因于吡啶氮原子质子化的pKa值为6.1，它与溶剂极性无关，而亚胺单质子化形式的pKa值从乙醇/水（3:17）中的12.5降至乙醇/水（49:1）中的11.3。将水解实验结果拟合到理论模型表明，存在一个pKa值为6.1的基团，它在水解动力学常数随pH值变化中起关键作用。对糖原磷酸化酶b的辅酶（磷酸吡哆醛）与羟胺反应活性的研究表明，该反应仅在溶液pH值低于6.5且亚胺键水解开始时发生。我们提出，当pH值小于6.2时磷酸化酶b活性的降低一定是由于酶 - 辅酶结合的断裂引起的，这可能与磷酸吡哆醛吡啶氮原子的质子化有关。
