MECHANISM-BASED INHIBITION OF THYMIDYLATE SYNTHASE BY 5-(TRIFLUOROMETHYL)-2'-DEOXYURIDINE 5'-MONOPHOSPHATE

Thymidylate synthase (TS) from Lactobacillus casei is inhibited by 5- (trifluoromethyl)-2'-deoxyuridine 5'-monophosphate (CF(3)dUMP). CF(3)d UMP binds to the active site of TS in the absence of 5,10-methylenetet rahydrofolate, and attack of the catalytic nucleophile cysteine 198 at C-6 Of the pyrimidine leads to activation of the trifluoromethyl grou p and release of fluoride ion. Subsequently, the activated heterocycle reacts with a nucleophile of the enzyme to form a moderately stable c ovalent complex. Proteolytic digestion of TS treated with [2'-H-3]CF(3 )dUMP, followed by sequencing of the labeled peptides, revealed that t yrosine 146 and cysteine 198 are covalently bound to the inhibitor in the enzyme-inhibitor complex. The presence of dithiothreitol (DTT) or beta-mercaptoethanol resulted in the breakdown of the covalent complex , and products from the breakdown of the complex were isolated and cha racterized. The three-dimensional structure of the enzyme-inhibitor co mplex was determined by X-ray crystallography, clearly demonstrating c ovalent attachment of the nucleotide to tyrosine 146. A chemical react ion mechanism for the inhibition of TS by CF(3)dUMP is presented that is consistent with the kinetic, biochemical, and structural results.