Covalent catalysis by pyridoxal: evaluation of the effect of the cofactor on the carbon acidity of glycine.
Covalent catalysis by pyridoxal: evaluation of the effect of the cofactor on the carbon acidity of glycine.
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DOI：
10.1021/ja0679228
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发表时间：
2007-03
影响因子：
15
通讯作者：
K. Tóth;J. Richard
中科院分区：
文献类型：
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作者：
K. Tóth;J. Richard
5'-脱氧吡哆醛(1)和甘氨酸之间的加合物的α -亚胺碳的去质子化的一级速率常数被确定为甘氨酸与1的claisen型加成的速率常数，其中去质子化是决定产物形成速率的速率常数。在pH 7.1下，1-甘氨酸铵离子的形态与碱态吡啶氮没有明显的去质子化。氢氧化物离子催化α -亚胺碳去质子化的kHO值分别从7.5 × 10(2)到3.8 × 10(5)到3.0 × 10(7) M(-1) s(-1)，分别随着吡啶氮、苯氧氧阴离子和1-甘氨酸亚胺离子的羧酸阴离子的质子化而增加。α -亚胺碳去质子化的pka相应从17降低到11，再降低到6。提出了酶选择性结合并催化与pKa = 17的反应。用HPO4（2-）催化α -亚胺碳去质子化的kB = 1.7 × 10(-3) s（-1）与丙氨酸消旋酶催化氨基酸消旋化的k(cat)/ k(m) = 4 × 10(5) m (-1) s（-1）进行比较，表明该酶能使α -亚胺碳去质子化的速率加快2 × 10(8)倍。这相当于丙氨酸消旋酶在催化丙氨酸去质子化过程中所承担的负荷的一半。
First-order rate constants for deprotonation of the alpha-imino carbon of the adduct between 5'-deoxypyridoxal (1) and glycine were determined as the rate constants for Claisen-type addition of glycine to 1 where deprotonation is rate determining for product formation. There is no significant deprotonation at pH 7.1 of the form of the 1-glycine iminium ion with the pyridine nitrogen in the basic form. The value of kHO for hydroxide ion-catalyzed deprotonation of the alpha-imino carbon increases from 7.5 x 10(2) to 3.8 x 10(5) to 3.0 x 10(7) M(-1) s(-1), respectively, with protonation of the pyridine nitrogen, the phenoxide oxyanion, and the carboxylate anion of the 1-glycine iminium ion. There is a corresponding decrease in the pKas for deprotonation of the alpha-imino carbon from 17 to 11 to 6. It is proposed that enzymes selectively bind and catalyze the reaction of the iminium ion with pKa = 17. A comparison of kB = 1.7 x 10(-3) s(-1) for deprotonation of the alpha-imino carbon of this cofactor-glycine adduct (pKa = 17 by HPO4(2-) with k(cat)/K(m) = 4 x 10(5) M(-1) s(-1) for catalysis of amino-acid racemization by alanine racemase shows that the enzyme causes a ca 2 x 10(8)-fold acceleration of the rate of deprotonation the alpha-imino carbon. This corresponds to about one-half of the burden borne by alanine racemase in catalysis of deprotonation of alanine.