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M, of 36,000 in the native and denatured forms. The hy- drolase is highly specific for (S)-3-hydroxyisobutyryl-. CoA and 3-hydroxypropionyl-CoA (K,,,,. 6 and 25 p ...

THEJOURNAL OF BIOLOGICAL CHEMISTRY 0 1994 by The American Society for Biochemistry and Molecular Biology, Inc.

Vol. 269, No. 19, Issue of May 13, pp. 14248-14253, 1994 Printed in U.S.A.

Purification and Partial Characterization of 3-Hydroxyisobutyryl-coenzymeA Hydrolase of Rat Liver” (Received for publication, August 30, 1993, and in revised form, February 21, 1994)

Yoshiharu ShimomuraSO, Taro Murakami8,Noriaki FujitsukaS, Naoya Nakain, Yuzo Saton, Satoru Sugiyamall, Noriko Shimomura**,Jamie IrwinSI, John W.HawesSS, and Robert A. Harris$+ From the $Department of Bioscience, Nagoya Institute of Technology, Showa-Ku, Nagoya 466, Japan and the $$Department of Biochemistry and Molecular Biology, Indiana University School of Medicine, Indianapolis, Indiana 46202-5122

An unusual feature of valine catabolism is a reaction in which an intermediate of its catabolic pathway, ( S ) 3-hydroxyisobutyryl-CoA, is hydrolyzed to give the free acid and CoA-SH. Theenzyme responsible for this reac(EC, tion,3-hydroxyisobutyryl-CoAhydrolase was purified 7200-fold from rat liver in this study. The purified enzymeconsists of asingle polypeptide with an M, of 36,000in the native and denatured forms.The hydrolase is highly specific for (S)-3-hydroxyisobutyrylCoA and 3-hydroxypropionyl-CoA(K,,,, 6 and 25 p ~ re, spectively) with optimal activity around pH 8. The turnoverrate of the enzymefor (S)-3-hydroxyisobutyryl-CoA is 270 s-l, which is high relative to other enzymes of the valine pathway. Likewise, activity of the enzyme expressed on a wet weightbasis is also very high in the major tissues of the rat. These findings suggest that rapid destruction of (S)-3-hydroxyisobutyryl-CoA producedduringvalinecatabolism is physiologically important. We propose thatthe need for a mechanism to protect cells against the toxic effects of methacrylylCoA, which is maintained in equilibrium with (S)-S-hydroxyisobutyryl-CoAby crotonase, explains why valine catabolism involves this enzyme and why its tissue activity is so high.

3-Hydroxyisobutyryl-CoA (HIB-CoA)’ hydrolase (3-hydroxy2-methylpropanoyl-CoA hydrolase, EC is responsible for the hydrolysis of (SI-HIB-CoA (11, an intermediate in the pathway of valine catabolism.This enzyme was partially purified from pig heartand some of its properties reported in1957 by Rendina and Coon (1).Purification of HIB-CoA hydrolase to homogeneity as well as further characterizationof some of its properties are reported here.The enzyme isof particular interest from the standpointof why the reaction it catalyzes should even occur in cells. A monocarboxylic acid is produced ((SI-3-

* This work was supported in part by National Institutes of Health Grant DK 40441 (to R. A. H.). The costs of publication of this article were defrayedin part by the payment of page charges. This article must therefore be hereby marked “advertisement” in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. 4 To whom correspondence should be addressed: Dept. of Bioscience, Nagoya Institute of Technology, Gokiso-Cho, Showa-Ku, Nagoya 466, Japan. Tel.: 81-52-732-2111 (ext. 2368); Fax: 81-52-732-9872. 1Present address: Research Center of Health, Physical Fitness and Sports, Nagoya University, Nagoya 464, Japan. 11 Present address: Inst. ofApplied Biochemistry, Gifu 505-01,Japan. ** Present address: Chukyo Junior College, Mizunami 509-61,Japan. The abbreviations used are: HIB-CoA, 3-hydroxyisobutyryl-CoA; PAGE, polyacrylamide gel electrophoresis; DTNB, 5,5‘-dithiobis(2-nitrobenzoicacid);Mes, 2-(N-morpholino)ethanesulfonicacid;CAPS, 3-(cyclohexylamino)propanesulfonicacid; Taps, 3-[tris(hydroxymethyl)methyllaminopropanesulfonic acid.

hydroxyisobutyricacid) that readily diffuses out of cells i n which i t is formed. Indeed, i t seems paradoxical that an acylCoA hydrolase should be required for this pathway when both proximal and distal parts of the pathway involve CoA ester intermediates. Furthermore, HIB-CoA hydrolase mustbe very specific for its substrate to avoid interference with catabolism of fatty acids, leucine,and isoleucine. Although therehas been much interest in the interorgan traffic of (S)-3-hydroxyisobutyrate and its possible roleas a substrate for various processes (2-61, we propose here that the reason for hydrolysis of (SIHIB-CoA is to protect cells against toxic effects of methacrylylCoA, an intermediate in the valine pathway occurring upstream of (S)-3-HIB-CoA. EXPERIMENTALPROCEDURES

Materials-For enzyme purification, livers were obtained from decapitated Sprague-Dawley rats that had been bred and raised in the laboratory. The tissue was stockpiled at -80 “C until sufficient quantities had accumulated. For tissue distribution studies, male Wistar rats were obtained from Harlan Industries (Indianapolis). Liver mitochondria were prepared by the method of Johnson and Lardy (7). Methacrylic anhydride, methacryloyl chloride, acryloyl chloride, and the Sand R-isomers of methyl-3-hydroxyisobutyratewere purchased from Aldrich; CoA-SH was purchased from Sigma or Kohjin Co., Ltd., Tokyo; crotonase, glutathione, glutathione-agarose, and thrombin were purchased from Sigma. Methacrylyl-CoA and acrylyl-CoA were prepared from methacrylic anhydride (or methacryloyl chloride) and acryloyl chloride, respectively, and CoA-SH according to the method of Stern and Campi110 (8) for the synthesis of crotonyl-CoA. Purification was accomplished by DEAE-cellulose chromatography as described by Lau et u1. (9). ( S ) -and (R)-HIB-CoA weresynthesized from the corresponding acids (obtained by alkaline hydrolysis of the (S)- and (R)-methyl esters) according tothe method of Wieland and Rueff (10).Purification was accomplished by reverse-phase high-performance liquid chromatography using an Applied Biosystems 130A separation system (220 x 2.1-mm C18 microbore column; 0-30% acetonitrile gradient). Both ( R ) and (SI-HIB-CoA eluted as single peaks a t 12.7% acetonitrile, gave single spots (R, = 0.69) on thin layer chromatography Avicel plates (Analtech Inc., Newark, DE) with a solvent system consisting of ethanoY0.l M sodium acetate, pH 4.5 (2:1, v/v),and reacted with NH,OH to give hydroxamates with the same R,(0.54) on thin layer chromatography (Avicel plates, H,O-saturated butanol as thesolvent system). The following CoA esters were obtained from Sigma: acetoacetyl-, acetyl-, benzoyl-, n-butyryl-, crotonyl-, glutaryl-, ~~-3-hydroxybutyryl-, 3-hydroxy-3-methylglutazyyl-, isobutyryl-, isovaleryl-,malonyl-, 3-methylcrotonyl-, DL-methylmalonyl-, palmitoyl-, phenylacetyl-, propionyl-, succinyl-, tiglyl-, and n-valezyl-. ( E ) -and (S)-ibuprofenyl-CoAwere kind gifts from Dr. Stephen D. Hall (Department of Medicine, Indiana University School of Medicine, Indianapolis, IN). Phenyl-Sepharose CL-4B, octyl-Sepharose CL-4B, DEAE-Sephacel, CM-Sepharose CL-GB, Sephacryl S-200HR, and thiopropyl-Sepharose 6B were purchased from Pharmacia LKBBiotechnology Inc.; hydroxylapatite (Bio-Gel HTP) was purchased fromBio-Rad; and ultrafiltration membrane Y”10 was purchased from Amicon, Inc. CoA-Sepharose wasprepared fromCoA-SH and thiopropyl-Sepharose 6B according to the instructions provided by Pharmacia. Recombinant 3-hydroxyisobutyratedehy-


Hydrolase 3-Hydroxyisobutyryl-CoA drogenase, expressed as a glutathione S-transferase fusion protein in Escherichia coli JM109 harboring a 3-hydroxyisobutyratedehydrogenase expression vector, was purified to homogeneityby glutathione-agarose affinity chromatography.z The fusion protein was cleaved with thrombin to give enzymeused for the quantitative assay of 3-hydroxyisobutyrate. All other reagents were analytical grade purchased from commercial sources. Composition of Bufers-Buffer A consisted of 50 mM potassium phosphate, pH 7.5,containing 0.1 mM EDTA, 0.1 p~ leupeptin, and 10 pg/ml trypsin inhibitor; Buffer B was 25 mM potassium phosphate, pH 7.5, containing 0.1 mM EDTA. Purification Procedures-All procedures were performed a t 0-4 "C. Frozen livers in 180-gportions were homogenizedwith an Oster blender (householdtype) at full speed for4 min in 720 mlof Buffer A containing 1 nm EDTA, 1%bovine serum, 10 N-tosyl-L-phenylalaninechloromethyl ketone, 0.1 mM phenylmethanesulfonyl fluoride, and 0.5% Triton X-100. The homogenate was centrifuged at 9500 x g for 15 min. The supernatant was passed through four layers of cheesecloth, and thepH was adjusted t o 7.5 with 2 M Tris. The supernatant was made to 30% saturation in ammonium sulfate (176 g/liter) by slow addition of a solid salt with constant stirring and was allowed to stir for 20 min before centrifugation at 9500 x g for 20 min. Fat was effectively removed by this step. The supernatant was passed through four layers of cheesecloth, made to 45% saturation in ammonium sulfate (addition of 94 g/liter) as above, and allowed to stir for 20 min before centrifugation at 9500 x g for 30min. The supernatant obtained was made to 75% saturation in ammonium sulfate (addition of 210 gfliter) as above and allowed to stir for 20 min beforecentrifugation at 9500 x g for 20 min. The pellet was dissolved in -100 ml of Buffer A and stored at -80 "C. The procedure described abovewas repeated once more. The preparations were thawed, combined, and applied to a phenylSepharose gel (350 ml) equilibrated with Buffer A containing 1 M ammonium sulfate on a sintered glass filter funnel (diameter, 9.5 cm). The gel was washed with the same buffer, and a fraction containing the g of HIB-CoA hydrolase activity not bound to the gel was collected. 332 solid ammonium sulfate (-80% final saturation) was slowly added to 870 ml of the fraction with constant stirring andfollowed by stirring for another 20 min before centrifugation at 9500 x g for 20 min. The pellet was dissolved in -60 ml of Buffer A andapplied to an octyl-Sepharose gel (-100 ml) equilibrated with Buffer A containing 1 M ammonium sulfate on a sintered glass filter funnel (diameter, 6.5 cm). The gel was washed with the same buffer, and a fraction containing the hydrolase activity not bound to the gel was collected and concentrated by ammonium sulfate precipitation as above. The pellet obtained was dissolved in -35 ml of 10 mM Tris-C1, pH 8.5, containing 0.1 mM EDTA and dialyzed against 1liter of the same buffer for 18 h with two buffer changes. After removal of aggregated proteins formed during dialysis by centrifugation (9500 x g for 10 min), the dialysate was applied to a DEAE-Sephacel column(2.5 x 15 cm) equilibrated with the samebuffer. The column was washed with the buffer at a flow rate of -65 ml/h until the absorbance of the eluate at 280 nm decreased almost to zero (-300 ml) and then eluted with 10 mM Tris-C1, pH 8.5, containing 0.1 mM EDTA and 0.1 M NaCl. The fractions with the hydrolase activity were combined and dialyzed against 2 liters of 10 nm K+/citratebuffer, pH 5.6, containing 0.1 mM EDTA overnight. After removal of aggregated proteins as above, the dialysate was applied to a CM-Sepharose column (2.5 x 8 cm) equilibrated with the same buffer. The column was washed with the buffer at a flow rate of -75 m l h until theabsorbance at 280 nmof the eluate was close to zero (-220 ml) and then eluted with 10 mM K+/citrate,pH 5.6, containing 0.1 mM EDTA and 0.1 M NaCl. The fractions with the hydrolase activity were combinedand concentrated to -5 ml by ultrafiltration (Y"10 membrane). The concentrate was diluted to -25 ml with Buffer B and again concentrated. This dilution and concentration cycle was repeated again to change buffer. The concentrate obtained was applied to a hydroxylapatite column (1.5 x 4 cm) equilibrated with Buffer B. The column was washed with the buffer at a flow rate of -20 ml/h until the absorbance of the eluate at 280 nm decreased almost to zero (-100 ml) and eluted with Buffer B containing 50 mM potassium phosphate (total 75 mM potassium phosphate). The main hydrolase activity fractions (Fig. 1)were combined, and Tween 20 was added to 0.01%. After concentration to -1.5 ml byultrafiltration, the preparation was made to 10%in glycerol and applied to a Sephacryl S-200 column (2.5x 46.5 cm) equilibrated with 50 mM potassium phosphate, pH 7.5, con-

'J. W. Hawes and R. A. Harris, unpublished method.


taining 0.1mM EDTA, 0.1 M KCl, and 0.05%Tween 20. The column was eluted with the buffer at a flow rate of 17.5 mlh. The fractions with the high ratio of the hydrolase activity t o absorbance at 280 nm (morethan 200) (Fig. 2) were combined and concentrated to -2 ml by ultrafiltration. The buffer of the preparation was changedto 10 mM K'lcitrate buffer, pH 5.6, containing 0.1 mM EDTA and 0.05% Tween 20 by the ultrafiltration method as above. The preparation was applied to a CoA-Sepharose column (1ml) equilibrated with the same buffer. The column was washed with 14 ml of the buffer at a flow rate of -30 ml/h and eluted with 10 nm K+/citratebuffer, pH 5.6, containing 0.1 mM EDTA, 0.05% Tween 20, and 0.1 M NaCl. The column was further eluted with the same buffer containing 0.5 M NaCl to remove proteins bound to the column and equilibrated again with 10 mM K+/citratebuffer, pH 5.6, containing 0.1 mM EDTA and 0.05% Tween 20. Fractions with the hydrolase activity eluted with the buffer containing 0.1 M NaCl were combined. The NaCl concentration was decreased to less than 10 mM by the ultrafiltration method, and the sample was applied again to the CoA-Sepharose column. The column was washed with 10 ml of the equilibrating buffer and eluted with Buffer B (pH 7.5) containing 0.05% Tween 20. The eluate was concentrated to -1.5 ml by ultrafiltration, made 10% in glycerol, and applied again to the Sephacryl S-200 columnas above. Protein peak fractions consisting of a single polypeptide (molecular weight of 36,000) examinedby SDS-PAGE were combinedand stored at -80 "C. Assay of Hydrolase Activity-The hydrolase activity was assayed spectrophotometrically at 30"C in a total volume of 1 ml with 0.1 M Tris-HC1, pH 8.0, containing 1 mM EDTA, 0.1% Triton X-100, 0.1 mM DTNB, 0.2 mM methacrylyl-CoA, and 10 units of crotonase. Approximately 1 min after addition of methacrylyl-CoA and crotonase, the reaction was started by addition of the indicated amount of the hydrolase dissolved in Buffer B containing 0.1% Tween20, and the absorbance change at 412 nm due to the reduction of DTNB (11)was monitored. The 3-hydroxypropionyl-CoAhydrolase activity was assayed in the same manner except that acrylyl-CoAwas used in place of methacrylyl-CoA. Oneunit of the hydrolase catalyzed the formation of 1 pmol of CoA-SWmin. Assay of HydrolaseActivity a t Variousp H Values-Areaction mixture in a total volume of 1ml with buffer (30 mM Mes, 30 mM Hepes, 30 mM Taps, 30 mM CAPS) containing 50 mM NaCI, 1 nm EDTA, 0.1% Triton X-100, 0.2 mM methacrylyl-CoA, and 10 units of crotonase was used 1 after adjustment to indicated pH with NaOH at 30 "C. Approximately min after addition of methacrylyl-CoAand crotonase, the reaction was started by the addition of 0.015 pg of the hydrolase followed by incubation at 30 "C for2.5 min. The reaction was stopped by the addition of 0.1 ml of a mixture of 18%trichloroacetic acid and 1%SDS. The pH of the mixture was adjusted to -8 by the addition of 1.5 M Tris-HCl, pH 8. Then, 0.1 ml of 1mM DTNB was added and absorbance at 412 nm was measured. A control mixture without the hydrolase was prepared and treated in the same manner. The absorbance obtained after enzyme reaction was corrected by the absorbance of control mixture. In the case of methylmalonyl-CoA (0.3mM) used as substrate, crotonase was omitted from the reaction mixture, 0.34 pgof the hydrolase was used, and the incubation for the reaction was performed at 30 "C for 40 min. Analytical Methods-3-Hydroxyisobutyrate was quantitated by the method of Rougraff et al. (12) with recombinant 3-hydroxyisobutyrate dehydrogenase as the enzyme source. SDS-PAGE was performed as described by Laemmli (13) except the acrylamide concentration was 12%.Samples for electrophoresis were treated as described previously (14).Protein determination was by the BCA method with bovine serum albumin as standard (15). RESULTS

Purification of HIB-CoAHydrolase from Rat Liuer-HIBCoA hydrolase was purified 7200-fold with an overall yield of 3.6% (Table I). Hydroxylapatite column chromatography proved to be one of the most effective steps of the purification with hydrolase activity being eluted with 75 mM phosphate buffer immediately after a major protein peak (Fig. 1). In the first Sephacryl S-200 column chromatography step (Fig. 2), it was found important to combine and recover only fractions with very high ratiosof hydrolase activity to absorbancea t 280 nm. Tween 20 had to be added t o all buffers after hydroxylapatite column chromatography tominimize loss of the enzyme by adsorption toplastic. The final preparation consisted of a single


Hydrolase 3-Hydroxyisobutyryl-CoA TABLEI Purification of HIB-CoAhydrolase from rat liver




Crude homogenate" (NH,),SO, (45-75'30) precipitate Phenyl-Sepharose and (NH,),SO, precipitate Octyl-Sepharose and (NH,)*SO, precipitate DEAEXephacel eluate CM-Sepharose eluate Hydroxylapatite eluate Sephacryl S-200 eluate CoA-Sepharose eluate Sephacryl S-200 eluate

Total activity


Specific activity





62,700 12,900 4,073 2,934 500 82.1 6.51 0.85 0.40 0.29

3,480 2,451 1,896 1,828 864 522 360 188 161 126

0.056 0.19 0.47 0.62 1.73 6.36 55.4 222 403 427

100 70 54 53 25 15

10 5.4 4.6 3.6

Refers to protein and activity of supernatant after centrifugation.

E 0


N c

m at 0 C


50 u)




Fraction number


Fraction number

FIG.2. Sephacryl 5-200 column chromatography. The preparaFIG.1. Hydroxylapatite column chromatography.The preparation obtained after CM-Sepharose column chromatography was subtion obtained after hydroxylapatite column chromatography was subjected to hydroxylapatite column chromatography.0, HIB-CoA hydro- jected to SephacrylS-200 column chromatography. 0, HIB-CoA hydroabsorbance a t 280 nm.Fractions 38 and 39 were absorbance a t 280 nm. The column was eluted with laseactivity; lase activity; Buffer B containing total potassium phosphate concentrationof 75 mM recovered for further purification. HIB-CoA hydrolase was eluted a t a (arrow A ) followed by Buffer B containing500 mM potassium phosphate molecular weight of 36,000. (arrow B ) . Fractions 2 2 4 4 were recovered for further purification.




polypeptide with a molecular weight of 36,000 as determined Mr by SDS-PAGE (Fig. 3). The molecular weight of the hydrolase was also 36,000 by gel filtration, indicating that the native enzyme is a monomer. 66k b pH Optimum and Lack of Cation a n d Nucleotide EffectsPartially purified HIB-CoA hydrolase was reported to have a 45kb pH of the assay pH optimum of about 5.6 (11,and that was the 36kb mixture used for the determination of activity in initial attempts to purify the enzyme. However, we found that the enzyme is active over a very wide range with theoptimum being 24kb about pH 8 (Fig. 4). This was not an artifact of the coupled assay since crotonaseis effective over a broad pH range and its activity at the concentrations used in the assay far exceeded D b that of HIB-CoA hydrolase. Thus, all subsequent work to purify FIG. 3. SDS-PAGE pattern of the purified HIB-CoA hydrolase. and characterize theenzyme was carried out at pH 8.0. 1 , 1 pg; lane 2, 2 pg; and lane 3, 4 pg of protein of the purified The following salts andnucleotides were without effect upon Lane HIB-CoA hydrolase. D refers to dye front. HIB-CoA hydrolase activity: 60mM KC1 and NaCl; 5 mM CaCI, and MgCI,; and 3 mM ATP, ADP, NAD+,and NADH. Substrate Specificity and Kinetics4S)-HIB-CoA, produced stereospecific for the S-isomer. Hydrolysis of the following comfrom methacrylyl-CoA by the action of crotonase, was the best pounds by the enzyme alsocould not be detected: acrylyl-, bensubstrate for HIB-CoA hydrolase under standard conditions zoyl-, n-butyryl-, crotonyl-, glutaryl-, 3-hydroxyisovaleryl- (pro(Table 11). 3-Hydroxypropionyl-CoA,produced from acrylyl-CoA duced from 3-methylcrotonyl-CoA by crotonase), 3-hydroxy-3by the action of crotonase, was also a good substrate. Ten other methylglutaryl-, (R)-ibuprofenyl-, (SI-ibuprofenyl-, isovaleryl-, CoA esters listed in Table I1 were hydrolyzed by the enzyme but 3-methylcrotonyl-, methacrylyl-, palmitoyl-, phenylacetyl-, sucat rates all less than1%of (S)-HIB-CoA. (R)-HIB-CoA was not cinyl-, and tiglyl-CoA. Thus, HIB-CoA hydrolase is highly spehydrolyzed by the enzyme, indicating HIB-CoA hydrolase is cific for the hydrolysis of (SI-HIB-CoA and 3-hydroxypropionyl-



Hydrolase 3-Hydroxyisobutyryl-CoA

pH Optimum for Methylmalonyl-CoA Hydrolysis by HIB-COA CoA. That the end products of the reaction catalyzedby HIBHydrolase-As noted above, DL-methylmalonyl-CoAwas hydroCoA hydrolasewith methacrylyl-CoA (pluscrotonase)as lyzed by HIB-CoA hydrolase, albeit slowly relative t o (SI-HIBsubstrate were indeed (S)-3-hydroxyisobutyrateand CoA-SH S )-methylmalonylwas confirmed by quantitating the amounts of each produced CoA (Table 11).It is interesting to note (that wasreported previously by under standard assay conditions. The ratio of (S)-3-hydroxy- CoA hydrolase(EC molecular isobutyrate toCoA-SH produced was 1.0 * 0.1 (mean* S.D. for Kovachy et al. (18, 19) to have almost the same weight (35,000) as found in the present study for HIB-CoA three separate determinations). For kinetic analysis, initial concentrations of (S)-HIB-CoA hydrolase. Optimal activity of (S)-methylmalonyl-CoA hydroand 3-hydroxypropionyl-CoA were calculatedfrom the extentof lase wasfound at pH 6 by Kovachy et al. (19), andthis was also their formationfrom methacrylyl-CoAand acrylyl-CoA at equi- found t o be the optimum pH for methylmalonyl-CoA hydrolysis librium by the hydration reactions catalyzed by crotonase. Ac- by HIB-CoA hydrolase (Fig. 4). cording to the optical assay described previously (17), 37% of Tissue Distribution of HZB-CoA Hydrolase-Distribution of methacrylyl-CoA and nearly 100% of acrylyl-CoA were con- HIB-CoA hydrolase in major rat tissues is given in Table IV. verted to their respective hydroxy-CoA esters within seconds Liver has the highest activity of the enzyme, followed closely by under the standard conditions of the HIB-CoA hydrolase assay. heart and then kidney. Muscle and brain have the lowest acLineweaver-Burk plots were linear with both substrates with tivities among the tissuesexamined. the lowest K,,, and turnover number being obtained with (SIThe HIB-CoA hydrolase activity of mitochondria prepared HIB-CoA (Table 111). from rat liver was found to be 152 milliunitdmg of protein. CoA esters that were hydrolyzed at slow rates inhibited (SI- Since liver contains -60 mg of mitochondrial proteidg, wet HIB-CoA hydrolysis competitively (Table 11). K, values were weight (20), this value fits well with the findingof an enzyme relatively high compared with theK,,, value for (SI-HIB-CoA. activity of -10 unitslg, wetweight, thereby confirming the view that HIB-CoA hydrolase is a mitochondrial enzyme. 500


HIB-CoA hydrolase has been purified to homogeneity from rat liver. The purified enzyme exhibits very high activity with 400 ( S)-HIB-CoAand 3-hydroxypropionyl-CoA as substrates.Activity was detectable with several short-chain CoA esters, but ( R1-HIB-CoA is not a substrate. No cofactors are necessaryfor 300 enzyme activity. The enzyme exists as a monomer with a molecular weight of 36,000. HIB-CoA hydrolase had beenpreviously purified %fold from 200 an alcohol-KC1 extract of pig heart (1). Amuch lower pH optimum for enzyme activity was reported for the partiallypurified enzyme (1)than found in the present study with thepurified 100 enzyme. The reasonfor the apparentdiscrepancy is not known, but a pH optimum slightly on the alkaline side makes good physiological sense considering the intracellular location of 0 HIB-CoA hydrolase (mitochondrial matrixspace). 5 6 7 8 9 1 0 HIB-CoA hydrolase shows great substratespecificity for ( S ) HIB-CoA and 3-hydroxypropionyl-CoA, thereby restricting its PH action to the valine catabolic pathway anda minor pathwayfor FIG.4.pH activityprofiles of HIB-CoAhydrolase with(S)-HIBCoA (0)and methylmalonyl-CoA(0) as substrates.The assaycon- propionate catabolism involving the latter CoA ester. The degree of specificity exhibited by the enzyme undoubtedly is imditions of enzyme activity are described under "Experimental Procedures." portant in preventing interference with numerous metabolic TABLEI1 Substrate specificity of HIB-CoAhydrolase and inhibition constants for various CoA esters CoA esters


Activity K,

Methacrylyl-CoA ((S)-HIB-CoA) + crotonase Acrylyl-CoA + crotonase (3-hydroxypropionyl-CoA) Tiglyl-CoA + crotonase (3-hydroxy-2-methylbutyryl-CoA) Crotonyl-CoA + crotonase (L-3-hydroxybutyryl-CoA) DL-3-Hydroxybutyryl-CoA 0.14 Acetoacetyl-CoA' 0.12 DL-Methylmalonyl-CoA A C I s o ~ u ~ ~ ~ Y ~ - C O 1.19 0.08 Malonyl-CoA Acetyl-coA Propionyl-CoA n-Valeryl-CoA



430" 244 3.14 1.75 1.15 0.94 0.64 0.34 0.26 1.60



m M


0.73 0.41

0.27 0.22 0.15


C* C C

0.06 C 0.04 2.61 C C 0.04 0.83 0.02 C The enzyme activity was assayed under standard conditions except that the concentrationsof CoA esters were at 0.5 mM, and crotonase was omitted for CoA esters other than those indicated. (5')-HIB-CoA For and 3-hydroxypropionyl-CoA, 0.011pg of enzyme protein was used, and for the other CoA esters 0.34 pg of enzyme protein was used. C, competitive. The valuesfor K , and type of inhibition were determinedby the method of Dixon and Webb (16). Acetoacetyl-CoA was used at a concentration of 0.2 mM. 0.17 0.17 0.72 0.09


3-Hydroxyisobutyryl-CoA TABLE 111 Kinetic characterization of HIB-CoA hydrolase








443 f 3 6.0 f 0.2 266

250 * 4 25 * 1.0 150

V,, (unitdmg)

K,,, (PMY Turnover number (s-’) Values were calculated from approximately 37and 100%conversion of methacrylyl-CoA to (SI-HIB-CoA and acrylyl-CoA to 3-hydroxypropionyl-CoA, respectively, a t equilibrium in the presence of crotonase.

Distribution of


TABLEIV HIB-CoA hydrolase in rat tissues



Liver Kidney Heart Muscle Brain f



CH3 Methacrylyl-CoA



HIB-COA hydrolase



\! \\

\\ \\ \\ \\ \\



9.9 f 0.8 6.2 5 0.3 8.3 f 0.3 2.0 f 0.4 2.7 f 0.1


S.E. for tissues from three animals.

Croronase I1 # CHZ=F-CSCoA


units fg, wet wt

“ HIB-CoA hydrolase activity was assayed under standard conditions. Each value is the mean

0 II



+Imbutyryl-CoA I

J 0 I1


0 II HC “ C H “0I

CH3 S-Methylmalonate semialdehyde-CoA

I4 II I1 11

CH3 processes involving CoA ester intermediates. Although the enS-Methylmalonate semialdehyde II zyme was found to hydrolyze nine additional CoA esters, all I1 were poor substrates relative to (S)-HIB-CoA. It is not likely MMSDH I1 that HIB-CoA hydrolase can hydrolyze any of these CoA esters & I Propionyl-CoA effectively under physiological conditions. However, their hy0 0 0 carboxylase I1 drolysis may be catabolized in the event that theirconcentraII II ATP 0 C CH CSCOA CH3-CH2CSCOA tion became markedly elevated due toa defect in a downstream I Propionyl-CoA enzyme of their catabolic pathway. The low but significant caCH3 S-Methylmalonyl-CoA pacity of HIB-CoA hydrolase to hydrolyze methylmalonyl-CoA is a case in point. Kovachy et al. (18, 19) have purified and partially characterized rat liver D(S)-methylmalonyl-CoA hySuccinyl-CoA drolase. This enzyme is notknown to havea function in normal metabolism, but rather it seems to be present in cells as a FIG.5. Valine catabolic pathway. HIBDH, 3-hydroxyisobutyrate safeguard to prevent CoA sequestration in the event of blockage dehydrogenase; MMSDH, methylmalonate semialdehyde dehydrogenof the propionyl-CoA catabolic pathway by an enzyme defect, ase. Dashed lines indicate hypothetical pathways. e.g. propionyl-CoA carboxylase deficiency or vitamin B,, deficiency. The characteristicsof the enzyme describedby Kovachy free acid under conditions wherethis CoA ester accumulates in et al. (18, 19) are quite similar to those of HIB-CoA hydrolase cells. with respect to the following: ( a ) active as monomer; ( b ) moIt is interesting to consider why a step is included in the lecular mass of 35,000 Da (compared with 36,000 Da for HIB- valine catabolic pathway (Fig. 5) in which a CoA ester is hyCoA hydrolase); ( c ) enzyme binding characteristics on anion- drolyzed to a free carboxylic acid. Destruction of a n activated and cation-exchange columns and CoA-Sepharose column; ( d l acyl group is rare in metabolic pathways, particularly when pH profile for the hydrolysis of methylmalonyl-CoA, ( e ) ster- subsequent steps of the pathway also involve activated intereospecificity with respect to S-isomer of CoA ester substrate; mediates. Since it seems that (S)-HIB-CoA could be converted and (f) enzyme specific activity with methylmalonyl-CoA as to (SI-methylmalonyl-CoA in just two steps (by a n alcohol desubstrate. Neither (SI-HIB-CoA nor 3-hydroxypropionyl-CoA hydrogenase followedby a n aldehyde dehydrogenase as dewere tested as substrates in the previous study of D(S)-meth- picted by dashed lines in Fig. 5) rather than the four steps ylmalonyl-CoA hydrolase (18,19).Although more definitive evi- actually usedby the pathway, including one that requiresATP, it seems odd that nature chose to sacrifice a CoA ester in the dence is needed, the present study suggests that the (SI-methylmalonyl-CoA hydrolaseactivity of the enzyme previously middle of what could have been a much simpler pathway. Howpurified by Kovachy et al. may correspond to a minor activityof ever, hydrolysis of (S)-HIB-CoA may be an important strategy for disposal of methacrylyl-CoA by cells. The lattercompound is HIB-CoA hydrolase. Hydrolysis of 2-methyl-3-hydroxybutyryl-CoAby HIB-CoA a thiol-reactive molecule that undoubtedly would inactivate hydrolase may occur under some conditions. This CoA ester is numerous enzymes in the absence of a mechanism designed to minimize its intramitochondrial concentration (22, 23). In formed by crotonase from tiglyl-CoA, a n intermediate in the isoleucine catabolic pathway. The normal pathway calls for a simpler pathway involving two dehydrogenases for direct conversion of 2-methyl-3-hydroxybutyryl-CoAto 2-methylace- conversion of (S)-HIB-CoA to (S)-methylmalonyl-CoA, the toacetyl-CoA. However, 2-methyl-3-hydroxybutyrate is found methacrylyl-CoA concentration would likely vary with themiin the urine of ketotic rats and humans (2), suggesting the tochondrial redox state, perhaps allowing toxic concentrations conditions latter conversion may be impeded by the redox state inketosis. of this thiol-reactive CoA ester to accumulate under Although HIB-CoA hydrolase hydrolyzes 2-methyl-3-hydroxy- of reducing equivalent overload. The clinical experience with an infant born with a n almost butyryl-CoA at a relatively slow rate (3 units/mg protein), it most likely is the enzyme responsible for the production of the complete deficiency of HIB-CoAhydrolase lends credence to the


3-Hydroxyisobutyryl-CoA above interpretation (22).The child exhibited multiple congenital physical malformations, suggesting that a defect in this enzyme may be teratogenic. Death from a cardiac lesion occurred at 3 months. During life, the patient excreted large amounts of cysteinekysteamine conjugates of methacrylic acid, indicating that conjugation between methacrylyl-CoA and glutathione occurred. Methacrylyl-CoA (but not the free acid) reacts readily with free thiol groups of proteins (22,231, suggesting that high concentrations could cause inhibition of enzymes with sensitive sulfhydryl groups. Methacrylate oxygen esters have been reported to be teratogenic (24) and are recognized as genotoxidclastogenic agents from studies with F344/N rats, B6C3F1 mice, and mouse lymphoma cells (25, 26). Because of the electron-withdrawing carboxylic acid oxygen ester group, compounds such as ethyl acrylate and methyl methacrylate (and the well known carcinogen acrylamide CH,=CHCONH,) readily react with the nucleophiles of proteins, DNA, and glutathione (Michael addition). Since thioesters are more electron-withdrawing than oxygen esters (271,we propose that methacrylyl-CoA and acrylyl-CoA are particularly reactive compounds with considerable potential forcytogenic, mutagenic, and clastogenic actions, making it important to maintain their intracellular concentrations extremely low. Acrylyl-CoA is a naturally occurring compound produced in small amounts from propionyl-CoA(1).Thus, it isinteresting to note that cells use the same strategyto protect against acrylylCoA toxicity as methacrylyl-CoA toxicity,i.e. conversion of acrylyl-CoA to 3-hydroxypropionyl-CoAby crotonase followed by thioester cleavage of the latter compound by HIB-CoA hydrolase to give the less reactive compound 3-hydroxypropionate. In contrastto the relatively low activities and turnover rates for the enzymes in distal and proximal parts of the valine pathway (activity and turnovernumbers of 1.2 pmol/midg, wet weight, of liver and 18 s-l for liver branched chain a-ketoacid dehydrogenase complex (14); 1.0 ymollmidg, wet weight, and 7 s-' for 3-hydroxyisobutyrate dehydrogenase (28);0.7pmol/ midg, wet weight, and 2 s-l for methylmalonate semialdehyde dehydrogenase (29)), HIB-CoAhydrolase has markedly higher tissue activity andturnover number (9.9pmol/min/g, wet weight, and 270 d ) , thereby accomplishing the rapid destruction of(5')-HIB-CoA as well as methacrylyl-CoA, the latter being due to the reaction catalyzed by crotonase, another enzyme with high tissue activity and turnover number. As a consequence, (S)-HIB-CoAand methacrylyl-CoA are not detectable in liver cells even when incubated under conditions that should maximize the concentrations of intermediates of the valine pathway (30). Formation of (S)-3-hydroxyisobutyric acid by the action of HIB-CoA hydrolase produces a carboxylic acidthat readily dif-



fuses from its intracellular site of formation. In some tissues, the enzymes that catalyze steps of the valine pathway distal to the reaction catalyzed by HIB-CoA hydrolase are quite poorly expressed, thereby establishing interorgan trafficking of (S)-3hydroxyisobutyrate (2-6, 21). Although there has been much interest in establishing a physiological role forcirculating (SI3-hydroxyisobutyrate, analogous to the important roles of circulating lactate and ketone bodies, its presence in blood may simply reflect the mechanism that has evolved to minimize methacrylyl-CoA toxicity. REFERENCES 1. Rendina, G., and Coon, M. J. (1957) J . Biol. Chem. 226, 523-534 2. Landaas, S. (1975) Clin. Chim. Acta 64, 143-154 3. Spydevold, 0.(1979) EUP.J. Biochem. 9 7 , 3 8 9 4 9 4 4. Wagenmakers, A. J. M., Salden, H. J. M., and Veerkamp, J. H. (1985) Int. J. Biochem. 17,957-965 5. Lee, S. H. C., and Davis, E. J. (1986) Biochem. J. 233,621-630 6. Letto, J., Brosnan, M. E., and Brosnan,J. T.(1986) Biochem. J. 240,909-912 7. Johnson, D., and Lardy, H. (1967) Methods Enzymol. 1 0 , 9 4 4 6 8. Stem, J. R., and Campillo, A. (1956) J. Biol. Chem. 218, 985-1002 9. Lau, E. P., Haley, B. E., and Barden,R. E.(1977) Biochemistry 16,2581-2585 10. Wieland, T., and Rueff, L. (1953)Angew. Chem. 66, 186187 11. Ellman, G. L. (1959) Arch. Biochem. Biophys. 82,70-77 12. Rougraff, P. M., Paxton, R., Goodwin, G . W., Gibson, R. G., and H a m s , R. A. (1990)Anal. Biochem. 184,317-320 13. Laemmli, U. K. (1970) Nature 227,680-685 14. Shimomura, Y.,Paxton, R., Ozawa, T., and Harris,R. A. (1987)Anal. Biochem. 163,7678 15. Smith, P. K., Krohn, R. I., Hermanson, G . T., Mallia, A. K., Gartner, F. H., Provenzano, M.D., Fujimoto, E. K., Goeke, N. M., Olson, B. J., and Klenk, D. C. (1985)Anal. Biochem. 160, 76-85 16. Dixon, M., and Webb, E. C. (1979) Enzymes, 3rd Ed., pp. 35S353, Longman Group, Ltd., London 17. Stem, J. R., Campillo, A., and Raw, I. (1956) J . Biol. Chem. 218,971-983 18. Kovachy, R., Copley, S. D., and Allen, R. H. (1983) J. Bid. Chem. 268,1141511421 19. Kovachy, R., Stabler, S. P., and Allen, R.H. (1988) Methods Enzymol. 166, 393-400 20. Zhang, B., Paxton, R., Goodwin, G . W., Shimomura, Y., and Hams,R. A. (1987) Biochem. J. 246,625-631 21. Kedishvili, N. Y.,Popov, K. M., Rougraff, P. M., Zhao, Y., Crabb, D. W., and H a m s , R. A. (1992) J. Biol. Chem. 267, 19724-19729 22. Brown, G. K., Hunt, M. S., Scholem, R., Fowler, K., Grimes, A,,Mercer, J . F. B., Truscott, R. M.,Cotton, R. G. H., Rogers, J. G., and Danks, D. M. (1982) Pediatrics 70, 532-538 23. Speir, T. W., and Bamsley, E. A. (1971) Biochem. J. 126,267-273 24. Singh, A. R., Lawrence, W. H., and Autian, J. (1972) J . Dent. Res. 61, 16321638 25. National Tbxicology Program Technical Report (1986) lbzicology and Carcinogenesis Studies ofMethyl Methacrylate in F344lN Rats and B6C3FlMice, NTP TR314, NIH Publication 87-2570, U. S. Public Health Service, Research Triangle Park, NC 26. Dearfield, K. L., Harrington-Brock, K., Doerr, C. L., Rabinowitz, J. R., and Moore, M. M.(1991) Mutagenesis 6, 519-525 27. Abeles, R. H., Frey, P. A,, and Jencks, W.P. (1992) Biochemistry, pp. 52-54, Jones and Bartlett,Boston 28. RougraE, P. M., Paxton, R., Kuntz, M. J., Crabb, D. W., and Hams,R. A.(1988) J. Biol. Chem. 263,327-331 29. Goodwin, G . W., Rougraff, P.M., Davis, E. J., and Harris,R. A. (1989)J. Biol. Chem. 264,14965-14971 30. Corkey, B. E., Martin-Requero, A,, Walajtys-Rode, E., Williams, R. J., and Williamson, J. R. (1982) J . Biol. Chem. 267, 9668-9676

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