KINETIC AND EQUILIBRIUM ALPHA-SECONDARY TRITIUM ISOTOPE EFFECTS AN REACTIONS CATALYZED BY DCMP HYDROXYMETHYLASE FROM BACTERIOPHAGE-T4

Deoxycytidylate (dCMP) hydroxymethylase (CH) catalyzes the formation o f 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 forma tion of a covalent complex containing enzyme, FdUMP, and CH2THF. Simil ar FdUMP complexes are formed by dTMP synthase and dUMP hydroxymethyla se, enzymes which are homologous to CH. Both the association and the d issociation 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 ef fect 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, sim ilar to that found for TS, indicate that C6 of the nucleotide is satur ated (i.e., sp(3) hybridized) in the covalent complex of CH, FdUMP, an d CH2THF. This strongly suggests that catalysis by CH proceeds via seq uential sp(2) --> sp(3) --> sp(2) hybridization changes at C6 of subst rate nucleotides, and it is consistent with a transient covalent linka ge 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 i s the expected inverse effect for sp(2) to sp(3) rehybridization of C6 either accompanying or preceding the first irreversible step in catal ysis by CH(D179N). The value of 1.0 for the observed KIE with dCMP ind icates that either (i) nucleophile addition to C6 occurs after the fir st irreversible step in catalysis or (ii) dissociation of the product Hm(5)dCMP from the enzyme is the first irreversible step of the reacti on. Either case indicates significant differences in the relative rate s of individual catalytic steps in the turnover of dCMP versus turnove r of dUMP, catalyzed by CH(D179N).