Spectroscopic detection of transient thiamin diphosphate-bound intermediates on benzoylformate decarboxylase

Thiamin diphosphate (ThDP)-dependent enzymes catalyze a range of transforma tions, such as decarboxylation and ligation. We report a novel spectroscopi c assay for detection of some of the ThDP-bound intermediates produced on b enzoylformate decarboxylase. Benzoylformate decarboxylase was mixed with it s alternate substrate p-nitrobenzoylformic acid on a rapid-scan stopped-flo w instrument, resulting in formation of three absorbing species (lambda (ma x) in parentheses): I-1 (a transient at 620 nm), I-1 (a transient at 400 nm ), and I-3 (a Stable absorbance with lambda (max) > 730 nm). Analysis of th e kinetics of the two transient species supports a model in which a noncova lent complex of the substrate and the enzyme is converted to the first cova lent intermediate I-1 the absorbance corresponding to I-1 is probably a cha rge-transfer band arising from the interaction of the thiamin diphosphate-p -nitrobenzoylformic acid covalent adduct (2-p-nitromandelylThDP) and the en zyme. The rate of disappearance of I-1 parallels the rate of formation of I -2. Chemical models suggest the lambda (max) of I-2 (near 400 nm) to be app ropriate to the enamine, a key intermediate in ThDP-dependent reactions res ulting from the decarboxylation of the thiamin diphosphste-p-nitrobenzoylfo rmic acid covalent adduct. Therefore, the rate of disappearance of I-1 and/ or the appearance of I-2 directly measure the rate of decarboxylation. A re laxation kinetic treatment of the pre-steady-state kinetic data also reveal ed a hitherto unreported facet of the mechanism, alternating active-sites r eactivity, Parallel studies of the His70Ala BFD active-site variant indicat e that it cannot form the complex reported by the charge-transfer band (I-1 ) at the level of the wild-type protein.