Role of tyrosine 65 in the mechanism of serine hydroxymethyltransferase
Role of tyrosine 65 in the mechanism of serine hydroxymethyltransferase
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DOI：
10.1021/bi000032z
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发表时间：
2000-06-27
期刊：
影响因子：
2.9
通讯作者：
Schirch, V
中科院分区：
文献类型：
--
作者：
Contestabile, R;Angelaccio, S;Schirch, V
人和兔胞浆丝氨酸羟甲基转移酶的晶体结构表明，Tyr 65可能是该酶作用机制中的关键残基。在大肠杆菌丝氨酸羟甲基转移酶与甘氨酸和5-甲酰四氢叶酸的三元复合物中，Tyr 65的羟基是底物甘氨酸的羧酸基团的氢键距离内的四个酶侧链之一。为了探测Tyr 65的作用，通过定点诱变将其改变为Phe 65。的Y 65 F位点突变体的三维结构进行了测定，并显示与野生型酶的同晶型，除了失踪的酪氨酸羟基。该突变酶在催化丝氨酸，甘氨酸，别苏氨酸，D-和L-丙氨酸，和5，10-亚甲基四氢叶酸底物的反应的动力学性质进行了测定。酶的性质与D-和L-丙氨酸，甘氨酸在没有四氢叶酸，和5，10-亚甲基四氢叶酸没有显着改变。然而，催化活性大大降低丝氨酸和别苏氨酸裂解和溶剂中的四氢叶酸的存在下，甘氨酸的Ct-质子交换。这些反应的催化活性降低可以解释为Y 65 F突变体丝氨酸羟甲基转移酶对这些氨基酸结合为外部醛亚胺的亲和力增加了2个数量级以上。这些数据与Tyr 65羟基在封闭活性位点转化为开放结构中的作用一致。
Crystal structures of human and rabbit cytosolic serine hydroxymethyltransferase have shown that Tyr65 is likely to be a key residue in the mechanism of the enzyme. In the ternary complex of Escherichia coli serine hydroxymethyltransferase with glycine and 5-formyltetrahydrofolate, the hydroxyl of Tyr65 is one of four enzyme side chains within hydrogen-bonding distance of the carboxylate group of the substrate glycine. To probe the role of Tyr65 it was changed by site-directed mutagenesis to Phe65. The three-dimensional structure of the Y65F site mutant was determined and shown to be isomorphous with the wild-type enzyme except for the missing Tyr hydroxyl group. The kinetic properties of this mutant enzyme in catalyzing reactions with serine, glycine, allothreonine, D- and L-alanine, and 5,10-methenyltetrahydrofolate substrates were determined. The properties of the enzyme with D- and L-alanine, glycine in the absence of tetrahydrofolate, and 5,10-methenyltetrahydrofolate were not significantly changed. However, catalytic activity was greatly decreased for serine and allothreonine cleavage and for the solvent ct-proton exchange of glycine in the presence of tetrahydrofolate. The decreased catalytic activity for these reactions could be explained by a greater than 2 orders of magnitude increase in affinity of Y65F mutant serine hydroxymethyltransferase for these amino acids bound as the external aldimine. These data are consistent with a role for the Tyr65 hydroxyl group in the conversion of a closed active site to an open structure.