Purification and characterization of 3-hydroxyisobutyrate dehydrogenase from rabbit liver

P. M. Rougraff, R. Paxton, M. J. Kuntz, David Crabb, Robert Harris

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Abstract

3-Hydroxyisobutyrate dehydrogenase (3-hydroxy-2-methyl propanoate: NAD+ oxidoreductase, EC 1.1.1.31) was purified 1800-fold from rabbit liver by detergent extraction, differential solubility in polyethylene glycol and (NH4)2SO4, and column chromatography on DEAE-Sephacel, phenyl-Sepharose, CM(carboxymethyl)-Sepharose, Affi-Gel Blue, and Ultrogel AcA-34. The enzyme had a native M(r) of 74,000 and appeared to be a homodimer with subunit M(r) = 34,000. The enzyme was specific for NAD+. It oxidized both S-3-hydroxyisobutyrate and R-3-hydroxyisobutyrate, but the k(cat)/K(m) was approximately 350-fold higher for the S-isomer. Steady state kinetic analysis indicates an ordered Bi Bi reaction mechanism with NAD+ binding before 3-hydroxyisobutyrate. The enzyme catalyzed oxidation of S-3-hydroxyisobutyrate between pH 7.0 and 11.5 with optimal activity between pH 9.0 and 11.0. The enzyme apparently does not have a metal ion requirement. Essential sulfhydryl groups may be present at both the 3-hydroxyisobutyrate and NAD+ binding sites since inhibition by sulfhydryl-binding agents was differentially blocked by each substrate. The enzyme is highly sensitive to product inhibition by NADH which may play an important physiological role in regulating the complete oxidation of valine beyond the formation of 3-hydroxyisobutyrate.

Original languageEnglish
Pages (from-to)327-331
Number of pages5
JournalJournal of Biological Chemistry
Volume263
Issue number1
StatePublished - 1988

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3-hydroxyisobutyrate dehydrogenase
Liver
NAD
Purification
Rabbits
Enzymes
Cibacron Blue F 3GA
Oxidation
Column chromatography
Propionates
Valine
Isomers
Detergents
Sepharose
Solubility
Metal ions
Chromatography
Oxidoreductases
Metals
Binding Sites

ASJC Scopus subject areas

  • Biochemistry

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Purification and characterization of 3-hydroxyisobutyrate dehydrogenase from rabbit liver. / Rougraff, P. M.; Paxton, R.; Kuntz, M. J.; Crabb, David; Harris, Robert.

In: Journal of Biological Chemistry, Vol. 263, No. 1, 1988, p. 327-331.

Research output: Contribution to journalArticle

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