The inactivation of thymidylate synthase by periodate.
Thymidylate synthase from methotrexate-resistant Lactobacillus casei rapidly lost about 90% of its catalytic activity when incubated with an equimolar concentration of IO4- at 0 degree C. Nearly complete inhibition resulted when the IO4- concentration was twice the enzyme concentration or higher. The inhibition reaction appeared to be pseudo-first-order with respect to enzyme when IO4- was in excess. The substrate dUMP, the product dTMP, and inorganic phosphate all protected the enzyme from inactivation by IO4-, with the order of effectiveness: dUMP greater than dTMP greater than phosphate. Deoxyuridine, which is not a substrate, did not protect the enzyme. Titrations with dithiobis(2-nitrobenzoate) (DTNB) showed that approximately 1.5 titratable SH groups were lost when thymidylate synthase was completely inhibited by IO4-. Essentially no reactivation occurred when periodate-inhibited enzyme was dialyzed against buffered 2-mercaptoethanol (ME) or dithiothreitol (DTT). Enzyme that had been treated with p-hydroxymercuribenzoate, DTNB, or methylmethanethiosulfonate prior to treatment with periodate could be completely reactivated with ME or DTT.
D003856:Deoxyuracil Nucleotides; D007780:Lacticaseibacillus casei / Q000201:enzymology; D008780:Methyltransferases / Q000037:antagonists & inhibitors*; D010504:Periodic Acid / Q000494:pharmacology*; D013438:Sulfhydryl Compounds; D013938:Thymidine Monophosphate; D013940:Thymidylate Synthase / Q000037:antagonists & inhibitors*
D003856:Deoxyuracil Nucleotides; D013438:Sulfhydryl Compounds; D010504:Periodic Acid; D013938:Thymidine Monophosphate; C007267:2'-deoxyuridylic acid; C009288:metaperiodate; D008780:Methyltransferases; D013940:Thymidylate Synthase