EVIDENCE FROM O-18 EXCHANGE STUDIES FOR AN EXOCYCLIC METHYLENE INTERMEDIATE IN THE REACTION CATALYZED BY T4 DEOXYCYTIDYLATE HYDROXYMETHYLASE

O-18 exchange experiments were designed to identify the final intermed iate in the catalytic mechanism of bacteriophage T4 deoxycytidylate (d CMP) hydroxymethylase (CH). CH catalyzes the formation of 5-(hydroxyme thyl)-dCMP (HmdCMP) from dCMP and methylenetetrahydrofolate (CH2-THF). CH resembles thymidylate synthase (TS), an enzyme of known three-dime nsional structure, in both amino acid sequence and the reaction cataly zed. 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 t o HmdCMP (by CH) or reduced to deoxythymidylate (by TS). We report her e that CH catalyzes the incorporation of O-18 from solvent water into the product, HmdCMP, in the presence of tetrahydrofolate (THF). The ca use of this exchange is a reverse reaction followed by a resynthesis. CH also catalyzes the exchange of O-18 from solvent water into HmdCMP in the absence of exogenous THF and in the presence of THF analogues t hat lack N-5. N-5 is the nitiogen that is likely to be bound to the me thylene as it is transferred to dCMP. A CH variant that lacks the nucl eophilic Cys 148 is incapable of promoting these O-18 exchange reactio ns. The THF analogues lacking N-5 do not promote a CH-catalyzed revers e reaction. Rather, we propose that the CH-catalyzed O-18 exchange rea ction promoted by these THF analogues occurs via 5-methylene-dCMP link ed to the enzyme through Cys 148. We conclude here that enzyme-bound 5 -methylene-dCMP is the final intermediate during catalysis by CH, as h as also een proposed for TS and dUMP.