Hardy L W, Nalivaika E
Department of Pharmacology, University of Massachusetts Medical Center, Worcester 01605.
Proc Natl Acad Sci U S A. 1992 Oct 15;89(20):9725-9. doi: 10.1073/pnas.89.20.9725.
The substrate preference of recombinant Escherichia coli thymidylate synthase (TS) has been altered from 2'-deoxyuridylate (dUMP) to 2'-deoxycytidylate (dCMP) by site-directed mutagenesis of the codon for Asn177, which was changed to aspartic acid. The side-chain amide of Asn177 forms hydrogen bonds with O4 and N3 of dUMP bound to the crystalline enzyme [Montfort, W. R., Perry, K. M., Fauman, E. B., Finer-Moore, J. S., Maley, G. F., Hardy, L., Maley, F. & Stroud, R. M. (1990) Biochemistry 29, 6964-6977]. This Asn is invariant in all natural sequences for TS known. The values of kcat for the mutant enzyme, TS(N177D), with dCMP and dUMP are, respectively, 0.09 and 0.002 times the value of kcat of wild-type TS with dUMP as substrate. TS(N177D) turns over dCMP at 35 times its rate of dUMP turnover, whereas wild-type TS turns over dCMP at < 10(-5) of its rate of dUMP turnover. Thus Asn177 is a major determinant of the pyrimidine nucleotide specificity of TS. The mutant enzyme, like wild-type TS, forms a covalent complex with 5-fluoro-dUMP in the presence of 5,10-methylenetetrahydrofolate. TS(N177D) also has a newly acquired ability to be transiently inactivated by dUMP. This time-dependent inactivation requires the presence of methylenetetrahydrofolate and may be due to the accumulation of the enzyme in the form of a catalytic intermediate. The likely mechanistic basis for discrimination by TS between dUMP and dCMP is their differing requirements for charge stabilization during covalent catalysis.
通过对天冬酰胺177（Asn177）密码子进行定点诱变，将其突变为天冬氨酸，已使重组大肠杆菌胸苷酸合成酶（TS）的底物偏好从2'-脱氧尿苷酸（dUMP）转变为2'-脱氧胞苷酸（dCMP）。Asn177的侧链酰胺与结合到晶体酶上的dUMP的O4和N3形成氢键[蒙特福特，W.R.，佩里，K.M.，福曼，E.B.，芬纳 - 摩尔，J.S.，马利，G.F.，哈迪，L.，马利，F. & 斯特劳德，R.M.（1990年）《生物化学》29卷，6964 - 6977页]。在已知的所有TS天然序列中，这个Asn是不变的。突变酶TS（N177D）以dCMP和dUMP为底物时的kcat值，分别是野生型TS以dUMP为底物时kcat值的0.09倍和0.002倍。TS（N177D）催化dCMP的周转速度是催化dUMP周转速度的35倍，而野生型TS催化dCMP的周转速度不到其催化dUMP周转速度的10^(-5)。因此，Asn177是TS嘧啶核苷酸特异性的主要决定因素。与野生型TS一样，突变酶在5,10 - 亚甲基四氢叶酸存在的情况下与5 - 氟 - dUMP形成共价复合物。TS（N177D）还获得了一种新特性，即会被dUMP瞬时失活。这种时间依赖性失活需要亚甲基四氢叶酸的存在，可能是由于酶以催化中间体的形式积累所致。TS区分dUMP和dCMP的可能机制基础是它们在共价催化过程中对电荷稳定化的不同要求。
