Kinetic and Equilibrium .alpha.-Secondary Tritium Isotope Effects on Reactions Catalyzed by dCMP Hydroxymethylase from Bacteriophage T4

Deoxycytidylate (dCMP) hydroxymethylase (CH) catalyzes the formation of 5-(hydroxymethyl)dCMP, essential for DNA synthesis in phage T4, from dCMP and methylenetetrahydrofolate (CH2THF). The nucleotide analog 5-fluorodeoxuridylate (FdUMP) stoichiometrically inactivates CH by formation of a covalent complex containing enzyme, FdUMP, and CH2THF. Similar FdUMP complexes are formed by dTMP synthase and dUMP hydroxymethylase, enzymes which are homologous to CH. Both the association and the dissociation rate of the FdUMP complex are shown to be increased by the mutation of active site Asp(179) to Asn. The mutated enzyme, CH(D179N), has an altered substrate preference, favoring dUMP rather than dCMP [Graves, K. L., et al. (1992) Biochemistry 31, 10315]. A value of 0.8 was determined for the alpha-secondary tritium equilibrium isotope effect on the binding of [6-H-3]FdUMP to wild-type CH and to CH(D179N), using a mixture of 2-C-14- and 6-H-3-labeled FdUMP. These effects, similar to that found for TS, indicate that C6 of the nucleotide is saturated (i.e., sp(3) hybridized) in the covalent complex of CH, FdUMP, and CH2THF. This strongly suggests that catalysis by CH proceeds via sequential sp(2) --> sp(3) --> sp(2) hybridization changes at C6 of substrate nucleotides, and it is consistent with a transient covalent linkage of C6 to the thiol of an essential CH residue, Cys(148). The values of the alpha-secondary H-3 kinetic isotope effect (KIE) on k(cat)/K-M for CH-catalyzed formation of Hm(5)dCMP caused by 6-H-3-substitution of dCMP, with both wild-type CH and CH(D179N), were very close to 1.0. However, the KIE for CH(D179N) with dUMP was 0.82. The latter value is the expected inverse effect for sp(2) to sp(3) rehybridization of C6 either accompanying or preceding the first irreversible step in catalysis by CH(D179N). The value of 1.0 for the observed KIE with dCMP indicates that either (i) nucleophile addition to C6 occurs after the first irreversible step in catalysis or (ii) dissociation of the product Hm(5)dCMP from the enzyme is the first irreversible step of the reaction. Either case indicates significant differences in the relative rates of individual catalytic steps in the turnover of dCMP versus turnover of dUMP, catalyzed by CH(D179N).