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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 kinetically at chemical equilibrium by monitoring radioisotope redistribution with sodium dl-hydroxy[3-(14)C]butyrate and [4-(3)H]NAD(+)(labelled in the nicotinamide ring). 2. When all substrates are maintained at concentrations approaching saturation (approx. 3-50 times the K(m) values) the first-order rate constant for the enzyme-catalysed interconversion of NAD(+) and NADH is much smaller than that for the enzyme-catalysed interconversion of d-3-hydroxybutyrate and acetoacetate. 3. The rate of interconversion of NAD(+) and NADH increases initially with increasing concentrations of d-3-hydroxybutyrate and acetoacetate (ratio of concentrations maintained constant), passes through a maximum and approaches closely to zero at saturating concentrations of the latter substrates. 4. The rates of interconversion of NAD(+) and NADH and of d-3-hydroxybutyrate and acetoacetate increase with increasing concentration of NAD(+) (up to 66 times its K(m) value) and NADH (up to 180 times its K(m) value) (ratio of the concentrations of the nicotinamide nucleotides maintained constant). 5. These findings support the description of this catalysis as an ordered Bi Bi mechanism with no detectable alternative pathway, in which the interconversion of the central ternary complexes is not rate-limiting, and provide no evidence for the formation of dead-end complexes. 6. The solubility of 2,4-dinitrophenylhydrazine in HCl exhibits an acidity optimum, the maximum solubility at 25.0 degrees C (3.8mg/ml, 19mm) occurring at 2.29m-HCl; in solutions of this acidity acetone 2,4-dinitrophenylhydrazone is relatively insoluble (0.098mg/ml, 0.413mm).
M J Preuveneers, and
D Peacock, and
E M Crook, and
J B Clark, and
K Brocklehurst
February 1967,
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M J Preuveneers, and
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K Brocklehurst
June 1968,
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K Brocklehurst
January 1977,
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M J Preuveneers, and
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K Brocklehurst
October 1972,
Biochimica et biophysica acta,
M J Preuveneers, and
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K Brocklehurst
September 1973,
FEBS letters,
M J Preuveneers, and
D Peacock, and
E M Crook, and
J B Clark, and
K Brocklehurst
September 1973,
Biochimica et biophysica acta,
M J Preuveneers, and
D Peacock, and
E M Crook, and
J B Clark, and
K Brocklehurst
September 1962,
Journal of general microbiology,
M J Preuveneers, and
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E M Crook, and
J B Clark, and
K Brocklehurst
January 2005,
FEMS microbiology letters,
M J Preuveneers, and
D Peacock, and
E M Crook, and
J B Clark, and
K Brocklehurst
August 1974,
Archives of biochemistry and biophysics,
M J Preuveneers, and
D Peacock, and
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J B Clark, and
K Brocklehurst
January 1975,
Archives of biochemistry and biophysics,
