Schirch D, Delle Fratte S, Iurescia S, Angelaccio S, Contestabile R, Bossa F, Schirch V
Department of Biochemistry and Molecular Biophysics, Virginia Commonwealth University, Richmond 23298.
J Biol Chem. 1993 Nov 5;268(31):23132-8.
Serine hydroxymethyltransferase has a conserved lysine residue (Lys-229) that forms the internal aldimine with pyridoxal 5'-phosphate. In other pyridoxal 5'-phosphate enzymes investigated so far, this conserved lysine residue also plays a catalytic role as a base that removes the alpha-proton from the amino acid substrate. Three mutant forms of Escherichia coli serine hydroxymethyltransferase (K229Q, K229R, and K229H) were constructed, expressed, and purified. The absorbance spectra, rapid reaction kinetics, and thermal denaturation of the mutant analogs were studied. Only the K229Q mutant serine hydroxymethyltransferase resembled the wild-type enzyme. The results indicate that Lys-229 plays a critical role in expelling the product by converting the external aldimine to an internal aldimine. In the absence of Lys-229, ammonia can also catalyze the same function at a much slower rate. However, Lys-229 apparently is not the base that removes the alpha-proton from the amino acid substrate. The K229Q mutant enzyme could catalyze one turnover of either serine to glycine or glycine to serine at rates approaching those of the wild-type enzyme. After one turnover, the mutant enzyme could not expel the product and bind new substrate. The K229Q mutant enzyme can also transaminate D-alanine, which, like the hydroxymethyltransferase activity, also requires removing the alpha-proton from the substrate. The absorbance spectra of the K229R and K229H serine hydroxymethyltransferases showed that their pyridoxal 5'-phosphate could not readily form an external aldimine with substrates, suggesting that Lys-229 in the wild-type enzyme may never bear a positive charge, further evidence that it is not the base that removes the alpha-proton.
丝氨酸羟甲基转移酶有一个保守的赖氨酸残基（Lys-229），它与磷酸吡哆醛形成内部醛亚胺。在迄今为止研究的其他磷酸吡哆醛酶中，这个保守的赖氨酸残基也作为碱发挥催化作用，从氨基酸底物上去除α-质子。构建、表达并纯化了大肠杆菌丝氨酸羟甲基转移酶的三种突变形式（K229Q、K229R和K229H）。研究了突变类似物的吸收光谱、快速反应动力学和热变性。只有K229Q突变型丝氨酸羟甲基转移酶与野生型酶相似。结果表明，Lys-229通过将外部醛亚胺转化为内部醛亚胺在排出产物中起关键作用。在没有Lys-229的情况下，氨也能以慢得多的速率催化相同的功能。然而，Lys-229显然不是从氨基酸底物上去除α-质子的碱。K229Q突变酶可以催化丝氨酸向甘氨酸或甘氨酸向丝氨酸的一次周转，速率接近野生型酶。一次周转后，突变酶无法排出产物并结合新的底物。K229Q突变酶也可以转氨D-丙氨酸，与羟甲基转移酶活性一样，这也需要从底物上去除α-质子。K229R和K229H丝氨酸羟甲基转移酶的吸收光谱表明，它们的磷酸吡哆醛不能轻易与底物形成外部醛亚胺，这表明野生型酶中的Lys-229可能从不带正电荷，进一步证明它不是去除α-质子的碱。
