title: Simultaneous Measurement of Glucose-6-phosphate 3-Dehydrogenase (NtdC) Catalysis and the Nonenzymatic Reaction of Its Product: Kinetics and Isotope Effects on the First Step in Kanosamine Biosynthesis.
abstract: Glucose-6-phosphate 3-dehydrogenase (NtdC) is an NAD-dependent oxidoreductase encoded in the NTD operon of Bacillus subtilis. The oxidation of glucose 6-phosphate by NtdC is the first step in kanosamine biosynthesis. The product, 3-oxo-d-glucose 6-phosphate (3oG6P), has never been synthesized or isolated. The NtdC-catalyzed reaction is very slow at low and neutral pH, and its rate increases to a maximum near pH 9.5. However, under alkaline conditions, the product is not stable because of ring opening followed by deprotonation of the 1,3-dicarbonyl compound. The absorbance band due to this enolate at 310 nm overlaps with that of the other enzymatic product, NADH, complicating kinetic measurements. We report the deconvolution of the resulting spectra of the reaction to determine the rate constants and likely kinetic mechanism. In doing so, we were able to determine the extinction coefficient of the enolate of 3oG6P (23000 M-1cm-1), which allowed the measurement of the first-order rate constant (5.51 × 10-3s-1) and activation energy (93 kJ mol-1) of nonenzymatic enolate formation. Using deuterium-labeled substrates, we show that hydride transfer from carbon 3 is partially rate-limiting in the enzymatic reaction, and deuterium substitution on carbon 2 has no significant effect on the enzymatic reaction but lowers the rate of deprotonation of 3oG6P 4-fold. These experiments clearly establish the regiochemistry of the reactions. Coupling of the NtdC reaction with the subsequent step in the pathway, NtdA-catalyzed glutamate-dependent amino transfer, has a small but significant effect on the rate of NAD reduction, consistent with these enzymes working together to process the unstable metabolite.
authors.affiliation: Department of Chemistry, University of Saskatchewan , 110 Science Place, Saskatoon, SK, Canada S7N 5C9.
authors.forename: Natasha D
authors.identifier: 0000-0002-0963-8858
authors.initials: ND
authors.lastname: Vetter
authors.affiliation: Department of Chemistry, University of Saskatchewan , 110 Science Place, Saskatoon, SK, Canada S7N 5C9.
authors.forename: David R J
authors.identifier: 0000-0001-9300-7460
authors.initials: DR
authors.lastname: Palmer
mesh_terms: D001412:Bacillus subtilis / Q000235:genetics / Q000378:metabolism*; D001426:Bacterial Proteins / Q000235:genetics / Q000378:metabolism*; D055162:Biocatalysis; D003903:Deuterium; D004926:Escherichia coli / Q000235:genetics / Q000378:metabolism; D015964:Gene Expression Regulation, Bacterial; D005944:Glucosamine / Q000096:biosynthesis; D019298:Glucose-6-Phosphate; D005954:Glucosephosphate Dehydrogenase / Q000235:genetics / Q000378:metabolism*; D018698:Glutamic Acid / Q000378:metabolism; D006863:Hydrogen-Ion Concentration; D007700:Kinetics; D009243:NAD / Q000378:metabolism; D009876:Operon; D010084:Oxidation-Reduction; D011522:Protons; D011994:Recombinant Proteins / Q000235:genetics / Q000378:metabolism; D013816:Thermodynamics
chemical_list: D001426:Bacterial Proteins; D011522:Protons; D011994:Recombinant Proteins; D009243:NAD; D018698:Glutamic Acid; D019298:Glucose-6-Phosphate; C014968:kanosamine; D003903:Deuterium; D005954:Glucosephosphate Dehydrogenase; D005944:Glucosamine