FUNCTIONAL-EFFECTS OF A NATURALLY-OCCURRING AMINO-ACID SUBSTITUTION IN HUMAN THYMIDYLATE SYNTHASE

A major mechanism underlying the cytotoxicity of fluoropyrimidine anal ogs such as 5-fluorouracil and 5-fluoro-2'-deoxyuridine (FdUrd) occurs via the formation of 5-fluoro-2'-deoxyuridylate (FdUMP), a tight-bind ing inhibitor of thymidylate synthase (TS). Genetic variation in the s tructure of the TS molecule is an important determinant of response to fluoropyrimidines, because such variation may affect the binding of F dUMP to the enzyme. Previous studies have shown that the colonic tumor cell line HCT116 expresses two structurally distinct TS polypeptides that differ by the presence of tyrosine or histidine at residue 33. Co mpared with the Tyr-33 form, the His-33 form confers a 3-4-fold level of FdUrd resistance to cells; this was postulated to be derived from t he reduced affinity of the enzyme for FdUMP and N5,N10)-methylenetetra hydrofolate, ligands required for the formation of a stable inhibitory complex. In the present study, the Tyr-33 and His-33 forms have been purified to homogeneity, and their properties have been compared in de tail. The K(m) values for dUMP and N5,N10-methylenetetrahydrofolate in the TS reaction were not significantly different between the two enzy mes. In contrast, the catalytic efficiency (k(cat)) was 8-fold lower f or the His-33 form. Kinetic and equilibrium binding measurements demon strated that the dissociation constant for FdUMP binding into the tern ary complex was 3-4-fold higher for the His-33 form; this was shown to be due to both a decrease in the rate of FdUMP association with the e nzyme and an increase in the rate of FdUMP dissociation from the terna ry complex. A TS form containing phenylalanine at residue 33 was creat ed by site-directed mutagenesis and was shown to be very similar to th e Tyr-33 enzyme with regard to k(cat), pH/activity profile, and effect on FdUrd response. Thus, it is the presence of histidine at residue 3 3, rather than the absence of tyrosine, that is responsible for the al terations in catalytic and ligand-binding functions exhibited by the H is-33 form. Possible mechanisms by which the histidine residue perturb s the structure of the TS active site are discussed.