GSBS Student Publications

Title

Kinetic and equilibrium alpha-secondary tritium isotope effects on reactions catalyzed by dCMP hydroxymethylase from bacteriophage T4

GSBS Program

Biochemistry & Molecular Pharmacology

UMMS Affiliation

Graduate School of Biomedical Sciences; Department of Pharmacology

Date

11-8-1994

Document Type

Article

Medical Subject Headings

Bacteriophage T4; Catalysis; Electrophoresis, Polyacrylamide Gel; Escherichia coli; Fluorodeoxyuridylate; *Hydroxymethyl and Formyl Transferases; Kinetics; Mutation; Protein Binding; Transferases; Tritium

Disciplines

Life Sciences | Medicine and Health Sciences

Abstract

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 Asp179 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 [6-3H]FdUMP to wild-type CH and to CH(D179N), using a mixture of 2-14C- and 6-3H-labeled FdUMP. These effects, similar to that found for TS, indicate that C6 of the nucleotide is saturated (i.e., sp3 hybridized) in the covalent complex of CH, FDUMP, and CH2THF. This strongly suggests that catalysis by CH proceeds via sequential sp2-->sp3-->sp2 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, Cys148. The values of the alpha-secondary 3H kinetic isotope effect (KIE) on kcat/KM for CH-catalyzed formation of Hm5dCMP caused by 6-3H-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.(ABSTRACT TRUNCATED AT 250 WORDS)

Rights and Permissions

Citation: Biochemistry. 1994 Nov 8;33(44):13049-56.

Related Resources

Link to article in PubMed

Journal Title

Biochemistry

PubMed ID

7947710