A Kinetic Study of Thymidylate Synthase from LactobaciZZus casei *

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.