Isolation and characterisation of the glycerol dehydrogenase from Bacillus stearothermophilus.
A protocol for the rapid purification of the glycerol dehydrogenase (glycerol: NAD+ 2-oxidoreductase, EC 1.1.1.6) from the thermophile Bacillus stearothermophilus has been developed using a combination of chromatographic techniques including affinity chromatography on a Sepharose-immobilised triazine dye (Procion red, HE3B, ICI). Substrate specificity has been examined and Km values determined. The protein has been shown to have an oligomeric Mr of approx. 180,000 and consists of four identical subunits of Mr 42,000. Exposure to chelating agents (e.g., EDTA) leads to total loss of activity; the EDTA-inactivated enzyme can be reactivated by Zn2+ and requires 1 mol equivalent of zinc per subunit for full catalytic activity. Other divalent cations such as Cd2+ and Co2+ will reactivate the apo-enzyme but yields an enzyme of lower specific activity. The enzyme binds 1 equivalent of NADH per subunit and during catalysis transfers the 4-pro-R hydride from the nicotinamide ring of the reduced-coenzyme to the substrate. Glycerol increases the dissociation constant for the interaction between NADH and Zn-metallo-glycerol dehydrogenase (ZnGDH) but has no effect on the equilibrium between NADH and metal-depleted enzyme.
Spencer
P
P
Department of Biochemistry, University of Southampton, U.K.
Bown
K J
KJ
Scawen
M D
MD
Atkinson
T
T
Gore
M G
MG
D000596:Amino Acids / Q000032:analysis; D002413:Cations, Divalent; D002845:Chromatography; D004098:Dihydroxyacetone / Q000378:metabolism; D004355:Drug Stability; D004492:Edetic Acid / Q000494:pharmacology; D004591:Electrophoresis, Polyacrylamide Gel; D004789:Enzyme Activation / Q000187:drug effects; D004793:Enzyme Reactivators; D001411:Geobacillus stearothermophilus / Q000201:enzymology*; D005990:Glycerol / Q000494:pharmacology; D046911:Macromolecular Substances; D008970:Molecular Weight; D009243:NAD / Q000378:metabolism; D013050:Spectrometry, Fluorescence; D013379:Substrate Specificity; D013401:Sugar Alcohol Dehydrogenases / Q000032:analysis / Q000302:isolation & purification / Q000378:metabolism; D015032:Zinc / Q000494:pharmacology
D000596:Amino Acids; D002413:Cations, Divalent; D004793:Enzyme Reactivators; D046911:Macromolecular Substances; D009243:NAD; D004492:Edetic Acid; D013401:Sugar Alcohol Dehydrogenases; C020672:glycerol dehydrogenase; D015032:Zinc; D004098:Dihydroxyacetone; D005990:Glycerol