| In 1973, Gutowski and Lienhard designed and synthesized thiamin thiazolone diphosphate (TTDP) as a transition state analogue for E. coli pyruvate dehydrogenase, an enzyme which uses thiamin diphosphate as a cofactor in the conversion of pyruvate to 2-(1-hydroxyethyl) thiamin diphosphate [Gutowski, J. A., and Lienhard, G. E. (1976) J. Biol. Chem., 251, 2863--2866]. Other groups have reported different results for other ThDP-dependent enzymes. Here, we begin the investigation of the interaction between TTDP and P. putida benzoylformate decarboxylase (BFD). First, a recombinant form of BFD was expressed and purified by nickel affinity chromatography. Existing direct and coupled assays were modified to follow the activity of the enzyme with endogenously bound thiamin diphosphate and inhibition of enzyme activity with TTDP. Preliminary results suggest K i = 5 muM for TTDP which is on the same order as the Km of ThDP for the enzyme. Thus, TTDP is not a transition state analogue for BFD. |
