Reiber H
Biochim Biophys Acta. 1976 Oct 22;444(3):734-55. doi: 10.1016/0304-4165(76)90321-4.
The overall reaction rates for the beta-elimination of serine and O-phosphoserine, catalyzed by various vitamin B-6 analogs (pyridoxal 5'-phosphate, 5'-deoxypyridoxal and N-methylpyridoxal 5'-phosphate) in the presence or absence of Cu2+ ions, are determined. The comparison of the pH-dependence of the molar activities of the three vitamin B-6 aldehydes in beta-elimination of serine enables the characterization of the different active Schiff base species and the single catalytic events. The Schiff base which has a positive charge on the pyridine ring nitrogen and a fully ionized phosphate group shows the highest molar activity. The phosphate group acts as an intramolecular general base catalyst, most probably at the alpha-carbon proton of the amino acid. Furthermore general acid catalysis by buffer species occurs at the beta-hydroxy group serine. These facts together provide a kinetically unambiguous description of the mechanism of the reaction: the removal of the proton at the alpha-carbon atom of serine is the rate-limiting step and is followed by the more rapid elimination of the b-hydroxy group of serine. The forward rate constant of the rate-limiting step is calculated for each of the reactions mentioned. The rate constants are compared with respect to the effectiveness of the individual catalytic components in the vitamin B-6-dependent beta-elimination. For optimal conditions the reaction of O-phosphoserine is faster by a factor of 10(4) in the velocity of the beta-elimination than the corresponding acid-catalyzed beta-elimination of serine. For the eliminations at the alpha- and beta-carbon atoms of O-phosphoserine in vitamin B-6-catalysed reactions a common transition state is discussed. From a comparison of the fastest vitamin beta-6-dependent model reaction with the rate of an enzymatic beta-elimination it is suggested that for those beta-elininating enzymes where the rate-limiting step is the same as in the model, the catalytic components mentioned could suffice to explain the velocity of the rate-limiting step.
测定了在有或没有Cu2+离子存在的情况下，各种维生素B-6类似物（磷酸吡哆醛、5'-脱氧吡哆醛和N-甲基磷酸吡哆醛）催化丝氨酸和O-磷酸丝氨酸β-消除反应的总体反应速率。通过比较三种维生素B-6醛在丝氨酸β-消除反应中摩尔活性对pH的依赖性，能够表征不同的活性席夫碱物种和单个催化事件。在吡啶环氮上带正电荷且磷酸基团完全电离的席夫碱表现出最高的摩尔活性。磷酸基团作为分子内通用碱催化剂，很可能作用于氨基酸的α-碳原子上的质子。此外，缓冲物种的通用酸催化发生在丝氨酸的β-羟基上。这些事实共同提供了对反应机理的动力学明确描述：丝氨酸α-碳原子上质子的去除是限速步骤，随后是丝氨酸β-羟基更快的消除。计算了上述每个反应限速步骤的正向速率常数。就维生素B-6依赖性β-消除中各个催化成分的有效性对速率常数进行了比较。在最佳条件下，O-磷酸丝氨酸的β-消除反应速度比相应的酸催化丝氨酸β-消除反应速度快10(4)倍。对于维生素B-6催化反应中O-磷酸丝氨酸α-和β-碳原子上的消除反应，讨论了一个共同的过渡态。通过比较最快的维生素β-6依赖性模型反应与酶促β-消除反应速率，表明对于那些限速步骤与模型相同的β-消除酶，上述催化成分可能足以解释限速步骤的速度。
