Costi P M, Liu L, Finer-Moore J S, Stroud R M, Santi D V
Department of Biochemistry and Biophysics, University of California, San Francisco, 94143-0448, USA.
Biochemistry. 1996 Apr 2;35(13):3944-9. doi: 10.1021/bi952642i.
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 2'-deoxyuridine 5'-monophosphate (dUMP). Asn 229 is not essential for substrate binding or catalysis [Liu, l., & Santi, D. B. (1993) Proc. Natl. Acad. Sci. U.S.A. 90, 8604-8608] but is a major determinant in substrate specificity [Liu, l., & Santi, D. V. (1993) Biochemistry 32, 9263-9267]. 3-Methyl-dUMP (3-MedUMP) is neither a substrate nor an inhibitor of wild type TS but is converted to 3-methyl 2'-deoxythymidine 5'-monophosphate by many TS Asn 229 mutants. Some of the Asn 229 mutants (N229C, -I, -M, -A, and -V) have kcat values for 3-MedUMP methylation which are up to about 20% of that for wild type TS-catalyzed methylation of dUMP, and some mutants (N229C and -A) catalyze methylation of 3-MedUMP more efficiently than that of dUMP. Mutants with hydrophobic side chains tended to be more active in catalysis of methylation of 3-MedUMP than those with hydrophilic side chains. The ability of 3-MedUMP to serve as a substrate for Asn 229 mutants shows that the active form of dUMP involves the neutral pyrimidine base and that ionization of the 3-NH group does not occur in the course of catalysis. In contrast to the negligible binding of 3-MedUMP to wild type TS, both 3-MedUMP and dUMP showed similar Km values with the Asn 229 mutants, suggesting similar binding affinities to the mutants. The X-ray crystal structure of the TS N229C--3-MedUMP complex showed that the side chain of Cys 229 was rotated away from the pyrimidine ring to allow placement of a water molecule and the 3-methyl group of 3-MedUMP in the active site. Our results suggest that the inability of 3-MedUMP to undergo methylation by wild type TS is due to its inability to bind to the enzyme, which in turn is simply a result of steric interference of the 3-methyl group with the side chain of Asn 229.
胸苷酸合成酶（TS）中保守的天冬酰胺229与核苷酸底物2'-脱氧尿苷5'-单磷酸（dUMP）的3-NH和4-O形成环状氢键网络。天冬酰胺229对于底物结合或催化并非不可或缺[刘林（Liu, l.）和桑蒂（Santi, D. B.）（1993年），《美国国家科学院院刊》90, 8604 - 8608]，但却是底物特异性的主要决定因素[刘林（Liu, l.）和桑蒂（Santi, D. V.）（1993年），《生物化学》32, 9263 - 9267]。3-甲基-dUMP（3-MedUMP）既不是野生型TS的底物，也不是其抑制剂，但许多TS天冬酰胺229突变体可将其转化为3-甲基2'-脱氧胸苷5'-单磷酸。一些天冬酰胺229突变体（N229C、-I、-M、-A和-V）对3-MedUMP甲基化的催化常数（kcat）值高达野生型TS催化dUMP甲基化kcat值的约20%，并且一些突变体（N229C和-A）催化3-MedUMP甲基化的效率高于催化dUMP甲基化的效率。具有疏水侧链的突变体在催化3-MedUMP甲基化方面往往比具有亲水侧链的突变体更具活性。3-MedUMP作为天冬酰胺229突变体底物的能力表明，dUMP的活性形式涉及中性嘧啶碱基，并且在催化过程中3-NH基团不会发生电离。与3-MedUMP与野生型TS的可忽略不计的结合相反，3-MedUMP和dUMP与天冬酰胺229突变体的米氏常数（Km）值相似，表明它们与突变体的结合亲和力相似。TS N229C - 3-MedUMP复合物的X射线晶体结构表明，半胱氨酸229的侧链从嘧啶环旋转开，以便在活性位点中放置一个水分子和3-MedUMP的3-甲基。我们的结果表明，野生型TS无法使3-MedUMP发生甲基化是由于其无法与该酶结合，而这反过来仅仅是3-甲基对天冬酰胺229侧链的空间干扰的结果。
