title: Structural, kinetic, and renaturation properties of an induced hydroxypyruvate reductase from Pseudomonas acidovorans.
abstract: A hydroxypyruvate reductase has been induced in Pseudomonas acidovorans by growth on glyoxylate. The enzyme has been purified to homogeneity as assessed by the criteria of analytical ultracentrifugation and analytical disc gel electrophoresis. It has a molecular weight of approximately 85,000 and is composed of two identical subunits. The subunits are not interconnected by disulfide bonds although the enzyme has 4 mol of half-cystine per mol of enzyme. The enzyme catalyzes the reversible conversion of hydroxypyruvate to D(minus)-glycerate in the presence of NADH. Glyoxylate cannot replace hydroxypyruvate as a substrate and is a competitive inhibitor of hydroxypyruvate reduction. The activity of the enzyme toward hydroxypyruvate is anion-modulated; the activity of the enzyme toward D(minus)-glycerate is unaffected by anions but is increased by tris-(hydroxymethyl)aminomethane. The subunits of the induced hydroxypyruvate reductase can be renatured. After the enzyme is dissociated in solutions of 6.0 M guanidine hydrochloride containing 0.1 M 2-mercaptoethanol, optimum renaturation occurs when subunits are diluted into a renaturation solvent consisting of 0.04 M Trischloride, pH 7.4, containing 25% glycerol, 25 mM 2-mercaptoethanol, and 0.14 MM NADH. NAD is an inhibitor of renaturation and therefore cannot substitute for NADH. The optimal temperature of dilution and subsequent incubation is 15 degrees, and increases in protein concentration up to 1.2 mg/ml, the highest concentration tested, improve both the rate of renaturation and the yield of active material. The half-time of renaturation at a protein concentration of 1.2 mg/ml was 1 min. The kinetics of renaturation is second order, i.e., is compatible with a bimolecular reaction preducted by the association of two similar subunits. The physical and kinetic parameters of the renatured protein are the same as those of the native enzyme.
mesh_terms: D000429:Alcohol Oxidoreductases / Q000378:metabolism*; D000596:Amino Acids / Q000032:analysis; D000838:Anions / Q000494:pharmacology; D004589:Electrophoresis, Disc; D005988:Glyceric Acids / Q000378:metabolism; D006146:Guanidines / Q000494:pharmacology; D006863:Hydrogen-Ion Concentration; D007700:Kinetics; D008623:Mercaptoethanol / Q000494:pharmacology; D008970:Molecular Weight; D009243:NAD / Q000378:metabolism; D009584:Nitrogen / Q000032:analysis; D011549:Pseudomonas / Q000201:enzymology; D011773:Pyruvates / Q000378:metabolism*; D013329:Structure-Activity Relationship; D013696:Temperature; D014461:Ultracentrifugation
chemical_list: D000596:Amino Acids; D000838:Anions; D005988:Glyceric Acids; D006146:Guanidines; D011773:Pyruvates; D009243:NAD; D008623:Mercaptoethanol; D000429:Alcohol Oxidoreductases; D009584:Nitrogen