Doumen J, Gonciarz M, Zegers I, Loris R, Wyns L, Steyaert J
Dienst Ultrastructuur, Vlaams Interuniversitair Instituut Biotechnologie, Vrije Universiteit Brussel, Sint-Genesius-Rode, Belgium.
Protein Sci. 1996 Aug;5(8):1523-30. doi: 10.1002/pro.5560050808.
The function of the conserved Phe 100 residue of RNase T1 (EC 3.1.27.3) has been investigated by site-directed mutagenesis and X-ray crystallography. Replacement of Phe 100 by alanine results in a mutant enzyme with kcat reduced 75-fold and a small increase in Km for the dinucleoside phosphate substrate GpC. The Phe 100 Ala substitution has similar effects on the turnover rates of GpC and its minimal analogue GpOMe, in which the leaving cytidine is replaced by methanol. The contribution to catalysis is independent of the nature of the leaving group, indicating that Phe 100 belongs to the primary site. The contribution of Phe 100 to catalysis may result from a direct van der Waals contact between its aromatic ring and the phosphate moiety of the substrate. Phe 100 may also contribute to the positioning of the pentacovalent phosphorus of the transition state, relative to other catalytic residues. If compared to the corresponding wild-type data, the structural implications of the mutation in the present crystal structure of Phe 100 Ala RNase T1 complexed with the specific inhibitor 2'-GMP are restricted to the active site. Repositioning of 2'-GMP, caused by the Phe 100 Ala mutation, generates new or improved contacts of the phosphate moiety with Arg 77 and His 92. In contrast, interactions with the Glu 58 carboxylate appear to be weakened. The effects of the His 92 Gln and Phe 100 Ala mutations on GpC turnover are additive in the corresponding double mutant, indicating that the contribution of Phe 100 to catalysis is independent of the catalytic acid His 92. The present results lead to the conclusion that apolar residues may contribute considerably to catalyze conversions of charged molecules to charged products, involving even more polar transition states.
通过定点诱变和X射线晶体学研究了核糖核酸酶T1（EC 3.1.27.3）中保守的苯丙氨酸100残基的功能。用丙氨酸取代苯丙氨酸100会产生一种突变酶，其催化常数（kcat）降低75倍，并且对于二核苷磷酸底物GpC的米氏常数（Km）略有增加。苯丙氨酸100突变为丙氨酸对GpC及其最小类似物GpOMe（其中离去的胞苷被甲醇取代）的周转速率有类似影响。对催化的贡献与离去基团的性质无关，表明苯丙氨酸100属于主要位点。苯丙氨酸100对催化的贡献可能源于其芳香环与底物磷酸部分之间直接的范德华接触。苯丙氨酸100也可能有助于过渡态五价磷相对于其他催化残基的定位。与相应的野生型数据相比，苯丙氨酸100突变为丙氨酸的核糖核酸酶T1与特异性抑制剂2'-鸟苷一磷酸（2'-GMP）复合的当前晶体结构中的突变的结构影响仅限于活性位点。由苯丙氨酸100突变为丙氨酸引起的2'-GMP的重新定位产生了磷酸部分与精氨酸77和组氨酸92的新的或改善的接触。相反，与谷氨酸58羧酸盐的相互作用似乎被削弱。在相应的双突变体中，组氨酸92突变为谷氨酰胺和苯丙氨酸100突变为丙氨酸对GpC周转的影响是累加的，表明苯丙氨酸100对催化的贡献独立于催化酸组氨酸92。目前的结果得出结论，非极性残基可能对催化带电分子转化为带电产物有很大贡献，甚至涉及更多极性的过渡态。
