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 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.
Gross
J W
JW
Department of Biochemistry, College of Agricultural Life Sciences, University of Wisconsin-Madison, Madison, Wisconsin 53705, USA.
Hegeman
A D
AD
Vestling
M M
MM
Frey
P A
PA
D001894:Borohydrides / Q000737:chemistry; D002384:Catalysis; D003903:Deuterium / Q000737:chemistry; D004795:Enzyme Stability; D004926:Escherichia coli / Q000201:enzymology; D005947:Glucose / Q000031:analogs & derivatives* / Q000737:chemistry; D006836:Hydro-Lyases / Q000737:chemistry*; D007700:Kinetics; D010084:Oxidation-Reduction; D019032:Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization / Q000379:methods; D013053:Spectrophotometry / Q000379:methods; D013329:Structure-Activity Relationship; D013379:Substrate Specificity; D013942:Thymine Nucleotides / Q000737:chemistry
D001894:Borohydrides; D013942:Thymine Nucleotides; C112447:dTDP-6-deoxy-4-ketoglucose; C103522:deoxythymidine diphosphate-glucose; C025364:sodium borohydride; D003903:Deuterium; D006836:Hydro-Lyases; C022771:dTDPglucose 4,6-dehydratase; D005947:Glucose