GSBS Student Publications

Title

Evidence from 18O exchange studies for an exocyclic methylene intermediate in the reaction catalyzed by T4 deoxycytidylate hydroxymethylase

GSBS Program

Biochemistry & Molecular Pharmacology

UMMS Affiliation

Graduate School of Biomedical Sciences

Date

8-30-1994

Document Type

Article

Medical Subject Headings

Bacteriophage T4; Catalysis; Deoxycytidine Monophosphate; Folic Acid; *Hydroxymethyl and Formyl Transferases; Kinetics; Magnetic Resonance Spectroscopy; Models, Chemical; Molecular Structure; Oxygen; Oxygen Isotopes; Tetrahydrofolates; Transferases

Disciplines

Life Sciences | Medicine and Health Sciences

Abstract

18O exchange experiments were designed to identify the final intermediate in the catalytic mechanism of bacteriophage T4 deoxycytidylate (dCMP) hydroxymethylase (CH). CH catalyzes the formation of 5-(hydroxymethyl)-dCMP (HmdCMP) from dCMP and methylenetetrahydrofolate (CH2-THF). CH resembles thymidylate synthase (TS), an enzyme of known three-dimensional structure, in both amino acid sequence and the reaction catalyzed. The final intermediate in the reaction catalyzed by TS or CH has been proposed to be the nucleotide with an exocyclic 5-methylene group covalently linked to the enzyme. This intermediate is then hydrated to HmdCMP (by CH) or reduced to deoxythymidylate (by TS). We report here that CH catalyzes the incorporation of 18O from solvent water into the product, HmdCMP, in the presence of tetrahydrofolate (THF). The cause of this exchange is a reverse reaction followed by a resynthesis. CH also catalyzes the exchange of 18O from solvent water into HmdCMP in the absence of exogenous THF and in the presence of THF analogues that lack N-5. N-5 is the nitrogen that is likely to be bound to the methylene as it is transferred to dCMP. A CH variant that lacks the nucleophilic Cys 148 is incapable of promoting these 18O exchange reactions. The THF analogues lacking N-5 do not promote a CH-catalyzed reverse reaction. Rather, we propose that the CH-catalyzed 18O exchange reaction promoted by these THF analogues occurs via 5-methylene-dCMP linked to the enzyme through Cys 148. We conclude here that enzyme-bound 5-methylene-dCMP is the final intermediate during catalysis by CH, as has also been proposed for TS and dUMP.

Rights and Permissions

Citation: Biochemistry. 1994 Aug 30;33(34):10521-6.

Related Resources

Link to article in PubMed

Journal Title

Biochemistry

PubMed ID

8068692