D-3-hydroxybutyrate dehydrogenase from Rhodopseudomonas spheroides. Kinetic mechanism from steady-state kinetics of the reaction catalysed by the enzyme in solution and covalently attached to diethylaminoethylcellulose.
1. The reversible NAD(+)-linked oxidation of d-3-hydroxybutyrate to acetoacetate in 0.1m-sodium pyrophosphate buffer, pH8.5, at 25.0 degrees C, catalysed by d-3-hydroxybutyrate dehydrogenase (d-3-hydroxybutyrate-NAD(+) oxidoreductase, EC 1.1.1.30), was studied by initial-velocity, dead-end inhibition and product-inhibition analysis. 2. The reactions were carried out on (a) the soluble enzyme from Rhodopseudomonas spheroides and (b) an insoluble derivative of this enzyme prepared by its covalent attachment to DEAE-cellulose by using 2-amino-4,6-dichloro-s-triazine as coupling agent. 3. The insolubilized enzyme preparation contained 5mg of protein/g wet wt. of total material, and when freshly prepared its specific activity was 1.2mumol/min per mg of protein, which is 67% of that of the soluble dialysed enzyme. 4. The reactions catalysed by both the enzyme in solution and the insolubilized enzyme were shown to follow sequential pathways in which the nicotinamide nucleotides bind obligatorily first to the enzyme. Evidence is presented for kinetically significant ternary complexes and that the rate-limiting step(s) of both catalyses probably involves isomerization of the enzyme-nicotinamide nucleotide complexes and/or dissociation of the nicotinamide nucleotides from the enzyme. Both catalyses therefore are probably best described as ordered Bi Bi mechanisms, possibly with multiple enzyme-nicotinamide nucleotide complexes. 5. The kinetic parameters and the calculable rate constants for the catalysis by the soluble enzyme are similar to the corresponding parameters and rate constants for the catalysis by the insolubilized enzyme.
D000090:Acetoacetates; D000244:Adenosine Diphosphate; D001665:Binding Sites; D002482:Cellulose; D002848:Chromatography, DEAE-Cellulose; D006884:Hydroxybutyrate Dehydrogenase / Q000037:antagonists & inhibitors; D006885:Hydroxybutyrates; D007700:Kinetics; D007773:Lactates; D008433:Mathematics; D008956:Models, Chemical; D009243:NAD; D009702:Nucleoside Diphosphate Sugars; D009904:Optical Rotation; D011485:Protein Binding; D012242:Rhodobacter sphaeroides / Q000201:enzymology; D012241:Rhodopseudomonas / Q000201:enzymology*; D012266:Ribose; D012995:Solubility; D013056:Spectrophotometry, Ultraviolet; D014227:Triazines
D000090:Acetoacetates; D006885:Hydroxybutyrates; D007773:Lactates; D009702:Nucleoside Diphosphate Sugars; D014227:Triazines; D009243:NAD; D000244:Adenosine Diphosphate; D012266:Ribose; D002482:Cellulose; D006884:Hydroxybutyrate Dehydrogenase