Liu L, Santi D V
Department of Biochemistry and Biophysics, University of California, San Francisco 94143-0448.
Biochemistry. 1992 Jun 9;31(22):5100-4. doi: 10.1021/bi00137a002.
The conserved Asn 229 of thymidylate synthase (TS) forms a cyclic hydrogen bond network with the 3-NH and 4-O of the nucleotide substrate dUMP. The Asn 229 to Asp mutant of Lactobacillus casei thymidylate synthase (TS N229D) has been prepared, purified, and investigated. Steady-state kinetic parameters of TS N229D show 3.5- and 10-fold increases in the Km values of CH2H4folate and dUMP, respectively, and a 1000-fold decrease in kcat. Most important, the Asp 229 mutation changes the substrate specificity of TS to an enzyme which recognizes and methylates dCMP in preference to dUMP. With TS N229D the Km for dCMP is bout 3-fold higher than for dUMP, and the Km for CH2H4folate is increased about 5-fold; however, the kcat for dCMP methylation is 120-fold higher than that for dUMP methylation. Specificity for dCMP versus dUMP, as measured by kcat/Km, changes from negligible with wild-type TS to about a 40-fold increase with TS N229D. TS N229D reacts with CH2H4folate and FdUMP or FdCMP to form ternary complexes which are analogous to the TS-FdUMP-CH2H4folate complex. From what is known of the mechanism and structure of TS, the dramatic change in substrate specificity of TS N229D is proposed to involve a hydrogen bond network between Asp 229 and the 3-N and 4-NH2 of the cytosine heterocycle, causing protonation of the 3-N and stabilization of a reactive imino tautomer. A similar mechanism is proposed for related enzymes which catalyze one-carbon transfers to cytosine heterocycles.
胸苷酸合成酶（TS）中保守的天冬酰胺229与核苷酸底物二氢尿嘧啶核苷酸（dUMP）的3 - NH和4 - O形成环状氢键网络。制备、纯化并研究了干酪乳杆菌胸苷酸合成酶（TS N229D）的天冬酰胺229突变为天冬氨酸的突变体。TS N229D的稳态动力学参数显示，二氢叶酸（CH2H4folate）和dUMP的米氏常数（Km）分别增加了3.5倍和10倍，而催化常数（kcat）降低了1000倍。最重要的是，天冬氨酸229突变将TS的底物特异性改变为一种优先识别并甲基化脱氧胞苷一磷酸（dCMP）而非dUMP的酶。对于TS N229D，dCMP的Km约比dUMP高3倍，CH2H4folate的Km增加了约5倍；然而，dCMP甲基化的kcat比dUMP甲基化的高120倍。通过kcat/Km测量，dCMP相对于dUMP的特异性从野生型TS时的可忽略不计变为TS N229D时增加约40倍。TS N229D与CH2H4folate和氟代脱氧尿苷一磷酸（FdUMP）或氟代脱氧胞苷一磷酸（FdCMP）反应形成三元复合物，这些复合物类似于TS - FdUMP - CH2H4folate复合物。根据已知的TS机制和结构，TS N229D底物特异性的显著变化被认为涉及天冬氨酸229与胞嘧啶杂环的3 - N和4 - NH2之间的氢键网络，导致3 - N质子化并稳定反应性亚氨基互变异构体。对于催化向胞嘧啶杂环转移一碳单位的相关酶，提出了类似的机制。
