Characterization of enzymatic processes by rapid mix-quench mass spectrometry: The case of dTDP-glucose 4,6-dehydratase

The single-turnover kinetic mechanism for the reaction catalyzed by dTDF-gl ucose 4,6-dehydratase (4,6-dehydratase) has been determined by rapid mix-ch emical quench mass spectrometry. Matrix-assisted laser desorption/ionizatio n time-of-flight mass spectrometry (MALDI-TOF MS) was employed to analyze q uenched samples. The results were compatible with the postulated reaction m echanism, in which NAD(+) initially oxidizes glucosyl C4 of dTDP-glucose to NADH and dTDP-4-ketoglucose, Next, water is eliminated between C5 and C6 o f dTDP-4-ketoglucose to form dTDP-4-ketoglucose-5,6-ene. Hydride transfer f rom NADH to C6 of dTDP-4-ketoglucose-5,6-ene regenerates NAD(+) and produce s the product dTDP-4-keto-6-deoxyglucose. The single-turnover reaction was quenched at various times on the millisecond scale with a mixture of 6 M gu anidine hydrochloride and sodium borohydride, which stopped the reaction an d reductively stabilized the intermediates and product. Quantitative MALDI- TOF MS analysis of the quenched samples allowed the simultaneous observatio n of the disappearance of substrate, transient appearance and disappearance of dTDP-hexopyranose-5,6-ene (the reductively stabilized dTDP-4-ketoglucos e-5,6-ene), and the appearance of product. Kinetic modeling of the process allowed rate constants for most of the steps of the reaction of dTDP-glucos e-d(7) to be evaluated. The transient formation and reaction of dTDP-4-keto glucose could not be observed, because this intermediate did not accumulate to detectable concentrations.