ASPARAGINE-229 IN THYMIDYLATE SYNTHASE CONTRIBUTES TO, BUT IS NOT ESSENTIAL FOR, CATALYSIS

The conserved Asn-229 (N229) of thymidylate synthase (TS, EC 2.1.1.45) provides the only side chain that directly hydrogen bonds with the py rimidine ring of the substrate dUMP. The carboxamide moiety forms a cy clic hydrogen bond network with the NH-3 and O-4 of the base and is a prime candidate for assisting proton-transfer reactions that occur at O-4 of the pyrimidine ring of dUMP. A complete replacement set of muta nts at position 229 of Lactobacillus casei TS (N229 mutants) has been prepared, purified, and characterized. Fifteen of the 19 TS mutants we re catalytically active. Steady-state kinetic parameters of N229 mutan ts varied 17- and 115-fold in the K(m) values for 5,10-methylene-5,6,7 ,8-tetrahydrofolate and dUMP, respectively, 1000-fold in k(cat) values , and 10,000-fold in k(cat)/K(m) values. Wild-type TS possesses lower K(m) and higher k(cat) and k(cat)/K(m) values than any of the TS N229 mutants. We conclude that N229 contributes to, but is not essential fo r, binding and catalysis. When the wild-type enzyme was not considered , there were excellent correlations between log k(cat) and the hydroph obicity of the side chains at position 229, in which the more hydropho bic side chains showed higher values. Our results suggest a unique int eraction between N229 and the substrates that seems important in appro priately positioning the uracil heterocycle for catalysis. We propose that in the absence of N229, the electrophilic catalyst that transfers protons to the O-4 and stabilizes enol intermediates is a highly cons erved molecule of water.