ASPARAGINE-229 MUTANTS OF THYMIDYLATE SYNTHASE CATALYZE THE METHYLATION OF 3-METHYL-2'-DEOXYURIDINE 5'-MONOPHOSPHATE

The conserved Asn 229 of thymidylate synthase (TS) forms a cyclic hydr ogen bond network with the 3-NH and 4-O of the nucleotide substrate 2' -deoxyuridine 5'-monophosphate (dUMP). Asn 229 is not essential for su bstrate binding or catalysis [Liu, L., gr Santi, D, V, (1993) Proc. Na tl. Acad. Sci, U.S.A, 90, 8604-8608] but is a major determinant in sub strate specificity [Liu, L., & Santi, D. V. (1993) Biochemistry 32, 99 63-9267], 3-Methyl-dUMP (3-MedUMP) is neither a substrate nor an inhib itor 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 k(cat) values for 3-MedUMP methylati on which are up to about 20% of that for wild type TS-catalyzed methyl ation of JUMP, 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 mon active in catalysis of methylation of 3- MedUMP than those with hydrophilic side chains. The ability of 3-MedUM P to serve as a substrate for Asn 229 mutants shows that the active fo rm 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 K-m values with the Asn 229 mutants, suggestin g similar binding affinities to the mutants. The X-ray crystal structu re 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 ill the active site. Our results suggest that the inability of 3-MedUMP to undergo methyla tion by wild type TS is due to its inability to bind to the enzyme, wh ich in turn is simply a result of steric Interference of the 3-methyl group with the side chain of Asn 229.