3-羟基异丁酸脱氢酶

Purification and Characterizationof 3-Hydroxyisobutyrate Dehydrogenase from Rabbit Liver*
(Received 87)
Paul M. Rougraff, Ralph PaxtonS, Martha Kuntz, DavidW. Crabb, and Robert A. Harris8 J. From the Departments of Biochemistry and Medicine, Indiana University School of Medicine, Indianqpolis,Indiana 46223 3-Hydroxyisobutyrate dehydrogenase (3-hydroxy- tural, kinetic, and regulatory features. In 1957, Robinson and 2-methyl propanoate: oxidoreductase, NAD+ EC Coon (1)reported on the characteristics of HIB dehydrogen1.1.1.31) was purified 1800-fold from rabbitliver by ase isolated from pig kidney. Pseudomonas aeruginosa (2) and detergent extraction, differential solubility in polyeth- Candida rugosa (3) enzymes have also been studied. Addiylene glycol and (NH4)%S04, column and chrotional characterization of mammalian HIB dehydrogenase is phenyl-Sepharose, matography on DEAE-Sephacel, of interest because 3-hydroxyisobutyrate increases in serum CM(carboxymethy1)-Sepharose,Affi-Gel Blue, and U1- and urine in ketoacidosis (4),and it is a good substrate for trogel AcA-34. The enzyme had a native Mr 74,000 hepatic gluconeogenesis (5). Furthermore, the exact pathways of and appeared to be a homodimer with subunit M, = for complete catabolism of the R- and S-isomersof 3-hydrox34,000. The enzyme was specific for NAD+. It oxidizedyisobutyrate remain uncertain for want of a description of the both S-3-hydroxyisobutyrate and R-3-hydroxyisobu- kinetics and substrate specificities of the enzymes involved tyrate, butthe kc.JKm was approximately 360-fold higher for the S-isomer. Steady state kinetic analysis (6). Recently, it was shown that HIB dehydrogenase is a major indicates an ordered Bi Bi reaction mechanism with contaminant in commercial preparations of RhodopseudomoNAD+ binding before 3-hydroxyisobutyrate. The enzyme catalyzed oxidation of S-3-hydroxyisobutyrate nas spheroides 3-hydroxybutyrate dehydrogenase (EC between pH 7.0 and 11.6 with optimal activity between 1.1.1.30; Ref. 7). Since the latter is used for enzymatic determination of serum ketone bodies @), serum and tissue 3pH 9.0 and 11.0. The enzyme apparently does not have hydroxybutyrate concentrations may have been significantly a metal ion requirement. Essential sulfhydryl groups may be present at both the 3-hydroxyisobutyrate and overestimated in numerous studies because of the contribution by 3-hydroxyisobutyrate. For this reason and because of NAD+binding sites since inhibitionbysulfhydryla binding agents w s differentially blocked by each sub- our limited understanding of the physiological regulation of strate. The enzymeis highly sensitive to product inhi- valine catabolism, it is important to determine 3-hydroxyisobition by NADH which may play an important physbutyrate concentrations in biological samples. Because HIB iological role in regulating the complete oxidation of dehydrogenase willprove useful for this purpose and for valine beyond the formation of 3-hydroxyisobutyrate. elucidating 3-hydroxyisobutyrate catabolism, we sought an inexpensive source and purification procedure for this enzyme. Rabbit liver is relatively rich in HIBdehydrogenase (1) and is commercially available in the frozen state. We report here the homogeneous purification and characterization of 3-Hydroxyisobutyrate dehydrogenase (3-hydroxy-2-meth- rabbit liver HIB dehydrogenase. ylpropanoate: NAD’ oxidoreductase, EC 1.1.1.31; HIB dehydrogenase)’ catalyzes an NAD’-dependent, reversible oxidaEXPERIMENTAL PROCEDURES’ tion of 3-hydroxyisobutyrate to methylmalonate semialdehyde. This mitochondrial enzyme, essential for valine catabolism, has not been well characterized with respect to strucRESULTS
* This work was supported in part by grants from the United States Public Health Service (NIH DK 19259 (to R.A.H.), AA 06434 and AA 00081 ( oD.W.C.)) and a short-term medical training grant (NIH t T35 HL7584 (to P.M.R.)); the Grace M. Showalter Residuary Trust and a Predoctoral Fellowship (to M.J.K) from the Indiana Heart Association. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked “advertisement” in accordance with 18U.S.C. Section 1734 solelyto indicate this fact. $ Present address: Dept. of Biological Sciences, Box 4149, Texas Tech University, Lubbock, T X 79409. 5 To whom correspondence and reprint requests should be addressed. ‘The abbreviations used are: HIB dehydrogenase (HIBDH in Miniprint), 3-hydroxyisobutyrate dehydrogenase (3-hydroxy-2-methylpropanoate:NAD+ oxidoreductase, EC 1.1.1.31); SDS-PAGE, sodium dodecyl sulfate-polyacrylamide gel electrophoresis; PEG, polyethylene glycol; DTT, dithiothreitol; Hepes, 4-(2-hydroxyethyl)1-piperazineethanesulfonic a c i d ; E G T A , [ e t h y l e n e b i s (oxyethylenenitrilo)]tetraaceticacid.
THE JOURNALBIOLOGICAL CHEMISTRY OF 0 1988 hy The American Society for Biochemistry and Molecular Biology, Inc.
VOl. 263, No. 1, Issue of January 5, pp. 327-331,1988 Printed in U. A. S.