A Kinetic Study of Thymidylate Synthase from LactobaciZZus

The kinetics of the reaction catalyzed by thymidylate synthase from Lactobacillus casei were investigated at pH 6.8 and 30” with the natural isomer of 5,10-methylene5,6,7&tetrahydrofolate ((+)-CH,-H,folate). An apparatus for adding the enzyme to the reaction mixture and rapidly mixing without opening the sample compartment allowed the acquisition of spectrophotometric rate data within a few seconds after initiating the reaction. Initial velocity data in the absence of products with phosphate buffer gave linear, intersecting double reciprocal plots consistent with a sequential mechanism. The inability to obtain similar plots with 1,4-piperazinediethanesulfonic acid buffer is ascribed to the lack of sufficient sensitivity for rate measurements at the low deoxyuridylate (dUMP) concentrations required. A double reciprocal plot of data when both substrates, in constant proportion, were simultaneously varied was parabolic and thus consistent with a sequential mechanism. Product inhibition by thymidylate (dTMP) was competitive when dUMP was the varied substrate and noncompetitive when (+I-CH,-H,folate was the varied substrate. ‘I,%Dihydrofolate (H,folate) inhibited noncompetitively with either substrate. The data support an ordered reaction sequence where dUMP adds to the enzyme before (+)-CH,H,folate and H,folate is released before dTMP. Michaelis constants for dUMP and (+)-CH,-H,folate were 0.70 FM and 14.0 PM, respectively. Dissociation constants of dUMP and dTMP from the binary enzyme complexes were 0.32 PM and 2.37 PM, respectively.