A rapid screen of active site mutants in glycinamide ribonucleotide transformylase
A rapid screen of active site mutants in glycinamide ribonucleotide transformylase
复制标题
DOI：
10.1021/bi9528715
复制
发表时间：
1996-07-09
期刊：
影响因子：
2.9
通讯作者：
Benkovic, SJ
中科院分区：
文献类型：
--
作者：
Warren, MS;Marolewski, AE;Benkovic, SJ
针对甘氨酰胺核糖核苷酸转移酶(EC 2.1.2.2)催化中心6埃以内的每个极性残基进行了定点突变和饱和定点突变。利用营养缺陷型细胞的功能互补，对这些突变体进行催化活性的快速筛选。通过这种筛选，可以快速定性地估计每个突变体的酶活性。这些结果表明，尽管Asn106、His108、Ser135和Asp144等几个极性残基对酶的全部催化活性非常重要，但靠近酶催化中心的极性残基没有一个是不可替代的。提出了一种固定的水分子介导底物和辅因子之间所需的质子转移，而10-甲酰基四氢叶酸通过甘氨酰胺核糖核酸胺的直接亲核攻击转移甲酰基的机理。活性中心的极性残基可以改变四面体中间体中进攻和离开氨基的pK(A)值，以促进甲酰基的转移。
Specific and saturation site-directed mutageneses have been used to alter each polar residue within 6 Angstrom of the catalytic center of glycinamide ribonucleotide transformylase (EC 2.1.2.2). These mutants were rapidly screened for catalytic activity using functional complementation of auxotrophic cells. This screen allows a rapid qualitative estimate of enzyme activity for each of these mutants. These results have shown that none of the polar residues close to the catalytic center of the enzyme are irreplaceable, although several are important for full catalytic activity, namely, Asn106, His108, Ser135, and Asp144. A mechanism is proposed in which a fixed water molecule mediates the required proton transfers between substrate and cofactor, while the formyl group is transferred from 10-formyltetrahydrofolate by direct nucleophilic attack by the amine of glycinamide ribonucleotide. The active site polar residues may act to alter the pK(a) values of the attacking and leaving amino groups within a putative tetrahedral intermediate in order to facilitate the transfer of the formyl group.