%0 Journal Article
%T Characterization of enzymatic processes by rapid mix-quench mass spectrometry: the case of dTDP-glucose 4,6-dehydratase.
%A Gross JW
%A Hegeman AD
%A Vestling MM
%A Frey PA
%J Biochemistry
%V 39
%N 45
%D Nov 2000 14
%M 11076501
%F 3.321
%R 10.1021/bi001963d
%X The single-turnover kinetic mechanism for the reaction catalyzed by dTDP-glucose 4,6-dehydratase (4,6-dehydratase) has been determined by rapid mix-chemical quench mass spectrometry. Matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS) was employed to analyze quenched samples. The results were compatible with the postulated reaction mechanism, in which NAD(+) initially oxidizes glucosyl C4 of dTDP-glucose to NADH and dTDP-4-ketoglucose. Next, water is eliminated between C5 and C6 of dTDP-4-ketoglucose to form dTDP-4-ketoglucose-5,6-ene. Hydride transfer from NADH to C6 of dTDP-4-ketoglucose-5,6-ene regenerates NAD(+) and produces 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 guanidine hydrochloride and sodium borohydride, which stopped the reaction and reductively stabilized the intermediates and product. Quantitative MALDI-TOF MS analysis of the quenched samples allowed the simultaneous observation of the disappearance of substrate, transient appearance and disappearance of dTDP-hexopyranose-5,6-ene (the reductively stabilized dTDP-4-ketoglucose-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-glucose-d(7) to be evaluated. The transient formation and reaction of dTDP-4-ketoglucose could not be observed, because this intermediate did not accumulate to detectable concentrations.