Interaction of thymidylate synthetase with 5-nitro-2'-deoxyuridylate.
5-Nitro-2'-deoxyuridylate (NO2dUMP) is a potent mechanism-based inhibitor of dTMP synthetase. After formation of a reversible enzymeìnhibitor complex, there is a rapid first order loss of enzyme activity which can be protected against by the nucleotide substrate dUMP. From studies of model chemical counterparts and the NO2dUMPdTMP synthetase complex, it has been demonstrated that a covalent bond is formed between a nucleophile of the enzyme and carbon 6 of NO2dUMP. The covalent NO2dUMPènzyme complex is sufficiently stable to permit isolation on nitrocellulose membranes, and dissociates to give unchanged NO2-dUMP with a first order rate constant of 8.9 x 10(-3) min-1. Dissociation of the complex formed with [6-3H]NO2dUMP shows a large alpha-secondary isotope effect of 19%, verifying that within the covalent complex, carbon 6 of the heterocycle is sp3-hybridized. The spectral changes which accompany formation of the NO2dUMPènzyme complex support the structural assignment and, when used to tritrate the binding sites, demonstrate that 2 mol of NO2dUMP are bound/mol of dimeric enzyme. The interaction of NO2dUMP with dTMP synthetase is quite different than that of other mechanism-based inhibitors such as 5-fluoro-2'-deoxyuridylate in that it neither requires nor is facilitated by the concomitant interaction of the folate cofactor, 5,10-CH2-H4folate, and that the covalent complex formed is unstable to protein denaturants.
D002250:Carbon Radioisotopes; D003856:Deoxyuracil Nucleotides / Q000494:pharmacology; D007553:Isotope Labeling; D007780:Lacticaseibacillus casei / Q000201:enzymology; D008780:Methyltransferases / Q000378:metabolism*; D011485:Protein Binding; D013056:Spectrophotometry, Ultraviolet; D013940:Thymidylate Synthase / Q000378:metabolism*; D014316:Tritium
D002250:Carbon Radioisotopes; D003856:Deoxyuracil Nucleotides; D014316:Tritium; C017563:5-nitro-2'-deoxyuridine 5'-monophosphate; D008780:Methyltransferases; D013940:Thymidylate Synthase