The inactivation of thymidylate synthase by periodate

Abstract

Thymidylate synthase from methotrexate-resistant Lactobacillus casei rapidly lost about 90% of its catalytic activity when incubated with an equimolar concentration of 10 4 − at 0°C Nearly complete inhibition resulted when the IO 4 − concentration was twice the enzyme concentration or higher. The inhibition reaction appeared to be pseudo-first-order with respect to enzyme when IO 4 − was in excess. The substrate dUMP, the product dTMP, and inorganic phosphate all protected the enzyme from inactivation by 10 4 −, with the order of effectiveness dUMP > dTMP > 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 10 4 −. 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-hydroxymercunbenzoate, DTNB, or methylmethanethiosulfonate prior to treatment with penodate could be completely reactivated with ME or DTT.