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2-Methylacetoacetyl-Coenzyme A reductase from Ascaris muscle: Purification and properties
ARCHIVES OF BIOCHEMISTRY AND BIOPHYSICS Vol. 226, No. 1, October 1, pp. 84-93, 1983
2-Methylacetoacetyl-Coenzyme Purification ZADILA
SUAREZ
DE
MATA, AND
A Reductase from Ascaris and Properties’
MARIA E. ZARRANZ, HOWARD J. SAZ*
RICHARD
Muscle: LIZARDO,
Departamento de Biologiu Celdar, Universidad Simon Bolivar, Apartah 80659, Camcu.s 1081A, Venezuela, and *Department of Biology, University of Notre Dame, Notre Dame, Indiana 46556 Received January
25, 1983, and in revised form April
29, 1983
2-Methylacetoacetyl-CoA and 3-keto-2-methyl pentanoyl-CoA have been proposed to be intermediates in the synthesis of 2-methylbutyrate and 2-methylvalerate, respectively, by Ascaris lumbricoides muscle. These volatile acids are major fermentation products of Asca& metabolism. 2-Methylacetoacetyl-CoA reductase has been purified 532-fold from Ascaris muscle to yield a homogeneous preparation which contained a single protein species as observed on discontinuous polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulfate. The purification procedure utilized subcellular fractionation, affinity chromatography on NAD+ agarose, and ion-exchange chromatography on DEAE-cellulose. A constant activity ratio for ethyl 2-methylacetoacetate and acetoacetyl-CoA was observed during purification, indicating that the same enzyme catalyzed both reactions. In addition, the purified protein catalyzed the NADH-dependent reduction of ethyl-3-keto-2-methyl pentanoate at essentially the same rate as it did ethyl 2-methylacetoacetate. The purified enzyme is a basic protein with an isoelectric point of 8.45 at 4°C. The molecular weight of the native protein (il4, = 64,000 by exclusion chromatography) and the size of the subunit (ilf, = 30,000 by dodecyl sulfate-polyacrylamide electrophoresis) indicate that the enzyme is composed of two subunits of the same molecular weight. Substrate-specificity studies, undertaken with the purified protein, demonstrated that the ethyl esters can substitute for the coenzyme A derivatives but this substitution results in an active substrate only when a branched 2-methyl group is present. The straight-chain ethyl ester is inactive. Kinetic constants for the substrates and nucleotides were determined. The role of the CoA esters as the physiological substrates for the Ascaris enzyme is substantiated. When assayed in the reductive direction with ethyl 2-methylacetoacetate as substrate, the activity of the purified enzyme was inhibited not only by coenzyme A as previously reported, but also by acetyl-CoA. The physiological implications of these inhibitions are discussed. ucts of carbohydrate metabolism by the parasitic intestinal roundworm, Ascaris lumbricaides var. suum (1). Only traces of n-butyrate, no n-hexanoate, and relatively minor quantities of n-valerate accumulate. This is in accord with the reports that Ascaris, like many other helminths, neither synthesizes long-chain fatty acids de nova nor catabolizes fatty acids appreciably. Isotope studies (2) and more recent enzymological investigations (3) indicate that the branched-chain volatile acids are formed by condensation of acetyl-CoA with
The branched-chain fatty acids, 2-methylbutyrate (2-MB)3 and 2-methylvalerate (2-MV), are the major fermentation prod1 This investigation was supported in part by Grants AI-09483 from the National Institutes of Health, U. S. Public Health Service, and Sl from Consejo National de Investigaciones Cientificas y Tecnologicas (CONICIT), Venezuela. * To whom reprint requests should be sent. a Abbreviations used: 2-M& 2-methylbutyrate; 2MV, 2-methylvalerate; Mes, 4-morpholineethanesulfonic acid. 0003-9861/83 $3.00 Copyright All rights
0 1983 by Academic Press, Inc. of reproduction in any form reserved.
84
Asumi
2-METHYLACETOACETYL-CoA
propionyl-CoA or by condensation of two propionyl-CoA molecules, respectively. The reactions involved are similar to, but in some important respects different from, a reversal of the B-oxidation pathway, particularly in regard to the corresponding enzymes of the host and the parasite. 2-Methylacetoacetyl-CoA, formed by the condensation of acetyl-CoA with propionylCoA, is subsequently reduced by 2-methylacetoacetyl-CoA reductase en route to the synthesis of 2-MB. Reduction of the carbony1 compound is NADH dependent and reversible. The reductive activity was localized in the soluble fraction of the Ascaris mitochondrion, and this activity has been employed to assay for the first reaction of the pathway, i.e., the condensation of the two acyl CoA units (3). The Ascaris enzyme is unique in that the apparent coenzyme A requirement can be substituted for by the ethyl esters of the substrates. However, the ethyl esters are active only if they contain a 2-methyl group. Although activity was reported with acetoacetyl-CoA when the soluble fraction of the Ascaris mitochondrion was the enzyme source (3), the possibility existed that this activity may be due to a different contaminating protein. It is of particular interest, however, that the pig heart L-3hydroxyacyl-CoA dehydrogenase differed from the Ascaris enzyme in that it was specific for the straight-chain CoA esters and no appreciable activity was observed with the branched-chain acyl CoA esters (3). Therefore, Ascaris enzyme serves physiologically almost exclusively for the reduction of the branched-chain compounds. The protein responsible for the reversible reduction of ethyl 2-methylacetoacetate from Ascaris mitochondria now has been purified and characterized. The purified enzyme is a basic protein comprised of two subunits of identical molecular weights. Results demonstrate that the same enzyme is responsible for the NADHdependent reduction of ethyl 3-keto2-methylbutyrate (ethyl methylacetoacetate), ethyl 3-keto-2-methylvalerate, acetoacetyl-CoA, and 3-keto-2-methylbutanoyl-S-thioglycolic acid. The enzyme also
85
REDUCTASE
catalyzed the reverse NAD+ dependent oxidation of ethyl 3-hydroxy-2-methylbutyrate, ethyl 3-hydroxy-2-methylvalerate, and 3-hydroxy-2-methylbutyryl-CoA. It is of particular interest that the Ascaris enzyme catalyzed the reduction reaction more rapidly, since the reduction would be required physiologically for the synthesis of 2-MB and 2-MV. MATERIALS
AND
METHODS
3-Hydroxy-2-methylbutyryl-CoA was synthesized from the mixed anhydride of (dl) 3-hydroxy-2-methylbutyric acid and ethyl chlorocarbonate (4). The mixed anhydride formed was assayed according to the procedure of Lipmann and Tuttle (7). Analysis of the acyl-CoA product by means of high-pressure liquid chromatography indicated that two main peaks were present in a 60~40 ratio. These comprised the two expected pairs of diastereoisomers (3). For the HPLC analysis, an aliquot of the chemically synthesized product was absorbed on a washed Sep-Pak Cl8 cartridge (Waters Associates, Inc. Milford, Mass.). Compounds of high polarity were removed by elution with water, and the acyl-CoA esters were eluted with 50% methanol. Aliquots containing the acyl-CoA ester were applied to a lo-pm Bondapak Cis column (4 mm X 30 cm). Separations were accomplished by elution with 43% methanol in water containing Pit A reagent (Waters Associates). 3-Keto-2-methylbutanoyl-S-thioglycolic acid was synthesized from ethyl 2-methylacetoacetate (Aldrich Chemical Co., Milwaukee, Wise.) by following the procedure described by Vagelos and Alberts (5) for the synthesis of 3-keto-octanoyl-9thioglycolic acid. Ethyl 3-keto-2-methylpentanoate (ethyl 3-keto-2methylvalerate) was synthesized by the condensation of ethyl propionate in the presence of sodium ethoxide (6). Ethyl 3-hydroxy-2-methylbutyrate was obtained by the reduction of ethyl methylacetoacetate (Aldrich) with potassium borohydride (3). Coenzyme A, acetyl-CoA, propionyl-CoA, acetoacetyl-CoA, and NAD+-hexane agarose (AGNAD, type 1) were purchased from P-L Biochemicals (Milwaukee, Wise.), and DEAE-cellulose (DE-52) was purchased from Whatman (Clifton, N. J.). Sephadex G-150, protein standards for molecular-weight determination, polybuffer exchange PBE 94, and polybuffer 96 were Pharmacia (Piscataway, N. J.) products. C,,-agarose employed for hydrophobic chromatography was purchased from Miles Laboratories (Elkhart, Ind.) Acyl-CoA esters were assayed according to Lipmann and Tuttle (7). Protein concentrations were determined by the Bradford method (8) or by the phenol reagent procedure (9) on samples treated with 1% (W/V) sodium dodecyl sulfate. Crystalline bovine
86
SUAREZ
DE MATA
serum albumin was used as standard with both methods. Mitochondria were prepared from dissected Ascaris muscle strips in 0.2 M sucrose, 5 mM EDTA, and 0.15% crystalline bovine serum albumin, pH 7.4, as described previously (10). 2-Methylacetoacetyl-CoA reductase was assayed spectrophotometrically at 340 nm by means of a modification of the method of Suarez de Mata et aL (3). The potassium phosphate buffer, pH 6.0, was replaced by 75 mM Mes, pH 5.5. Unless otherwise stated, the substrate employed for assay was ethyl 2-methylacetoacetate. Activity in the reverse (oxidative) direction was measured spectrophotometrically at 340 nm by following the concomitant reduction of NAD+ as described by Suarez de Mata et al (3). One unit of enzyme activity is equivalent to one micromole of substrate transformed per minute. Electrophoresis of protein was carried out on polyacrylamide slab gels in the presence of 0.1% sodium dodecyl sulfate with the discontinuous buffer system and gel formulation of Laemmli (11). Samples were denatured prior to electrophoresis by heating at 100°C for 30 min in 1% sodium dodecyl sulfate containing 1% mercaptoethanol.
Pur$ication of .%Methylacetoacetyl-CoA Reductase 1. Isolation of mitochundrial soluble fraction. 2Methylacetoacetyl-CoA reductase activity is localized and was isolated from the soluble fraction of the Ascoris mitochondrion (3). Washed mitochondria obtained from 60 g (wet wt) of adult female Ascaris muscle were resuspended to 27 ml with 0.02 M potassium phosphate buffer, pH 6.0. This suspension was freeze-thawed and subsequently sonicated in the cold with 10 pulses of 30 s each with 60-s cooling intervals in a Biosonic III sonicator, fitted with a microtip at a power setting of 30 W (Bronwill Scientific, Rochester, N. Y.) The mitochondrial soluble fraction was obtained after centrifugation at 269,000g for 30 min. 2. Ammonium sulfate precipitation Sufficient solid ammonium sulfate was added to the mitochondrial soluble fraction to make the solution 80% saturated (51.6 g/200 ml) (12). After standing for 30 min at 4”C, the mixture was centrifuged at 13,500g for 30 min. The yellow precipitate obtained was made to 3 ml with 0.02 M potassium phosphate buffer, pH 6.0. The sample then was desalted by passage over a column (1.5 x 5 cm) of Sephadex G-50 equilibrated with 20 rnM potassium phosphate buffer, pH 6.0. 3. NAD+-Agarose a&nity chromatography. The above sample was applied to an NAD+-agarose column (0.9 X 8 cm) equilibrated with 20 mM potassium phosphate buffer, pH 6.0. After a wash with 20 ml of the equilibration buffer, elution was carried out with 20 ml of a 2.6 mM NADH solution in 20 mM potassium
ET AL.
phosphate buffer, pH 8.0. Fractions of 1.0 ml were collected and the enzymatically active fractions were pooled. 4 DEAE-ceUulcse and hydrqdnbic chromatography. The active fractions eluted from the NAD+-agarose column were applied to a DEAE-cellulose column (0.2 X 21 cm) which was equilibrated with 20 mM potassium phosphate buffer, pH 7.5. Elution of the column was carried out with 20 ml of the equilibration buffer. Only occasionally was a minor contaminant present in this eluate, as indicated by electrophoresis on sodium dodecyl sulfate-acrylamide slab gels. Only when this contaminant was present was the additional step added. This contaminant was eliminated by subjecting the sample to hydrophobic chromatography on a Cl0 column (0.9 X 3.5 cm) equilibrated in the above buffer. The column was washed sequentially with 8 ml each of equilibration buffer containing 0 and 1.0 M NaCl, and eluted with 10 ml of 1.0 M NaCl in equilibration buffer containing 50% ethylene glycol. The sample eluted had a slightly higher specific activity and contained 78% of the initial activity. When necessary for gel chromatography, DEAEcellulose and Cl0 active fractions were concentrated by ultrafiltration using YM-10 Diaflo membranes (Amicon Corp., Danvers, Mass.). The isoelectric point of the Ascaris 2-methylacetoacetyl-CoA reductase was determined by chromatofocusing on a column (0.9 X 30 cm) of polybuffer exchanger PBE 94 equilibrated with 0.025 M ethanolamine-acetic acid buffer, pH 9.2, and eluted with polybuffer 96 adjusted to pH 6.0 with 1.0 M acetic acid.
RESULTS
Enzyme Pur$cation 2-Methylacetoacetyl-CoA reductase was purified from the soluble fraction of the Ascaris mitochondrion by means of affinity chromatography on NAD+-agarose and ion-exchange chromatography on DEAEcellulose. The purified enzyme had a specific activity of 55 units/mg of protein (Table I) after a 538-fold purification at a yield of 24%. The value of 538-fold purification was calculated by taking into consideration that the isolation of the mitochondrial soluble fraction from crude homogenates resulted in a 19-fold purification in terms of 2-methylacetoacetyl-CoA reductase activity. It should be emphasized that at the NAD+-agarose step both propionyl-CoA condensing enzyme and enoyl-CoA hydratase activities are separated from the reductase. These activities, which also are
Ascuri.s
2-METHYLACETOACETYL-CoA TABLE
I
PURIFICATIONOF Ascaris 2-METHYLACETOACETYL-COA 2-Methylacetoacetyl-CoA
Fraction 1. Mitochondrial soluble” 2. Ammonium sulfate (O-80%) 3. NAD-Agarose 4. DEAE-cellulose
87
REDUCTASE
REDUCTASE
reductase activity
Specific activity ratio: Ethyl methylacetoacetate/ Acetoacetyl-CoA
Total protein (md
Total (units)
47.5
92.2
1.9
100
1
2.68
32.6 3.16 0.4
78.4 44.5 22.3
2.4 14.1 55.1
85 48 24
1.2 7.3 28.3
3.2 2.6 3.0
a Isolation of the mitochondrial terms of 2-methylacetoacetyl-CoA
Specific activity (units/mg protein)
Percentage recovery
soluble fraction from crude homogenate reductase activity.
involved in the synthesis of 2-MB and 2MV, did not bind to NAD+-agarose and were recovered in the wash of the column. The composition of the fractions at the various stages of the purification was followed by dodecyl sulfate-polyacrylamide gel electrophoresis (Fig. 1). The final preparations were homogeneous on these gels, a single protein band being observed. This band was present in approximately equal intensity when protein amounts corresponding to equal amounts of reductase activity were applied to the gel and, presumably, would be derived from the active enzyme.
Fold purification
resulted in a Is-fold
purification
in
The isoelectric point of the Ascaris enzyme was determined by chromatofocusing, as described under Materials and Methods. The value obtained by this method was equal to a pH of 8.45. This pH value was consistent with the observed behavior of the enzyme on DEAE-cellulose. This technique also served as an additional criterion for homogeneity, since when the purified preparation was first applied to the same column, but equilibrated with 0.025 M imidazole HCl buffer, pH 7.4, and eluted with polybuffer 74 set to pH 4.0 (data not shown), only one symmetrical protein peak which overlapped with the activity peak was observed.
Chamcterization of LMethylacetoacetyG CoA Reductase.
Substrate Specificity of the Reductase
The molecular weight of the purified Ascaris 2-methylacetoacetyl-CoA reductase was determined chromatographically on a column (1 X 43 cm) of Sephadex G-150, eluted with 10 mM potassium phosphate buffer, pH 7.5. The enzyme eluted at a position corresponding to M, 64,000.The same results were obtained employing an AcA 34 Ultrogel column (1 X 40 cm) equilibrated with 20 mM potassium phosphate buffer, pH 7.5. The number and size of the subunit(s) in the native protein were investigated by means of sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The dissociated enzyme formed a single protein band with an electrophoretic mobility corresponding to M, 30,000.
The HPLC spectrum of the synthetic 3-hydroxy-2-methylbutyryl-CoA showed two major peaks in a 60:40 ratio. These peaks comprised the two expected pairs of diastereoisomers. Initial rates of oxidation of 3-hydroxy-2-methylbutyryl-CoA and the reduction of 3-keto-2-methylbutanoyl-Sthioglycolate, respectively, were determined at increasing concentrations of each substrate (Figs. 2 and 3). Normal Michaelis-Menten responses were observed with these synthetic compounds. Comparative substrate-specificity studies were examined (Table II). The ethyl esters of both 2methylacetoacetate and 3-keto-2-methylpentanoate, the presumed precursors of 2MB and 2-MV, were reduced at essentially the same rate by the purified enzyme. No
88
SUAREZ
DE MATA
ET AL.
when a branched methyl group is present since no activity could be detected on ethyl acetoacetate, whereas the CoA ester of acetoacetate did show activity. The chemically synthesized thioglycolate ester of 3keto-2-methylbutyrate was reduced by the purified enzyme at a rate approaching that obtained with the ethyl ester. The reductase reaction was reversible, but the ethyl esters of both 3-hydroxy-2methylbutyrate and 3-hydroxy-2-methylvalerate were oxidized at approximately one-twentieth the rate of the reverse, reduction reaction. The difference between the rate of the two reactions became somewhat less when the coenzyme A ester of 3-hydroxy-2-methylbutyrate was employed as substrate for the oxidative reaction, but remained approximately one-tenth that of the reduction reaction. This is of particular interest, since physiologically the enzyme would function primarily in the faster, reductive direction. The purified reductase was specific for NAD+. Neither NADP+ nor NADPH was utilized. 1
234567
8
9
10
FIG. 1. Purification of 2-methylacetacetyl-CoA reductase by discontinuous gel electrophoresis in 0.1% sodium dodecyl sulfate. A 9% acrylamide running gel with 3% stacking gel was formulated and run as described under Materials and Methods. The contents of each slot were 1, reference proteins; 2, mitochondrial soluble fraction; 3, ammonium sulfate pellet; 4, NAD-agarose fraction; 5, DEAE-cellulose fraction; 6,7, and 8, CIO-hydrophobic fraction; 9, reference proteins; 10, chymotrypsinogen. To slots 2 through 6 was added an aliquot from the respective purification step containing 0.025 units of reductase activity. Slots 7 and 8 contained 0.13 and 0.08 units, respectively.
activity was observed with the ethyl ester of acetoacetate or with the lithium salt of methylacetoacetate, confirming the requirement for both the branched methyl and ester groups. Acetoacetyl-CoA was reduced by the purified enzyme, but only at approximately one-third the rate of the ethyl esters of the branched-chain acids. In addition, a constant activity ratio of ethyl methylacetoacetate/acetoacetyl-CoA reduction was observed during purification (Table I). It appears that the ethyl ester can substitute for the CoA derivative only
Kinetic Constants of the Ascaris Reductase The Km values obtained with Ascaris reductase were approximately one order of magnitude higher when the ethyl esters were employed as substrates, compared with the CoA derivatives (Table III). Most important, the Km for 3-hydroxy-2-methylbutyryl-CoA was 33 times lower than that for the corresponding ethyl ester and compared more favorably with the values obtained for the physiological nucleotides, NADH and NAD+. The V,,, values obtained for both the ethyl and thioglycolate esters of 2-methylacetoacetate were approximately the same, and three times higher than the V,,, obtained for acetoacetyl-CoA. In addition, more than a twofold increase in the I’,,,, was observed with the CoA ester of 3-hydroxy-2-methylbutyrate in comparison to the corresponding ethyl ester. Neither monovalent nor divalent cations had an appreciable effect upon the purified Ascaris ethyl 2-methylacetoacetate reduc-
Asmti
2-METHYLACETOACETYL-CA
89
REDUCTASE
0.7
0.6
0.5
f \ a
0.4
0
0.3
0.2
0.1
1
0
02 [b-Hydroxy-2-
I
0.4
L
0.6
methylbutyryl
0.6
t
IO
-I 1.;2
COA] mM.
FIG. 2. Dependence of the initial rate of 3-hydroxy-2-methylbutyryl-CoA oxidation on substrate concentration. Assay conditions were as described under Materials and Methods. Assays contained 2.8 fig of protein. Substrate concentrations were determined by the method of Lipmann and Tuttle (7). I
I
6 /
[J-K&o
-2-mathylbuionoyl-S-
thioglycolic
acid ] mht
FIG. 3. Dependence of the initial rate of the reduction of 3-keto-2-methylbutanoyl-S-thioglycolic acid on substrate concentration. Assay conditions were as described under Materials and Methods. Assay contained 3.4 pg of protein. Substrate concentrations were determined by the method of Lipmann and Tuttle (7).
90
SUAREZ TABLE
DE MATA
Eflect of Acetyl-CoA on b Methylacetoacetyl-CoA Reductase Activity
II
SUBSTRATE SPECIFICITY OF PURIFIED Ascaris 2-METHYLACETOACETYL-CoA REDUCTASE Activity” (pmol/min/ mg protein)
Substrate Propionyl-CoA Ethyl 2-methylacetoacetats Ethyl 3-keto-2-methylpentanoate Ethyl Acetoacetate Lithium salt of methylacetoacetate Acetoacetyl-CoA 3-Keto-2-methylbutanoyl-Sthioglycolic acid Ethyl 3-hydroxy-2-methylvalerate + NAD+ Ethyl 3-hydroxy-2-methylbutyrate + NAD+ 3-Hydroxy-2-methylbutyryl-CoA + NAD+
ET AL.
0 49.1 51.0 0 0 16.2 37.0 3.4 2.0 5.0
a Enzyme activities were determined spectrophotometrically as described under Materials and Methods. The molar concentrations of the substrates employed were propionyl-CoA, 2 X 10e3 M; ethyl 2-methylacetoacetate, 11.7 X 10-s M; ethyl 3-keto-2-methylpentanoate, 11.7 X 10-s M; ethyl acetoacetate, 11 X 10e3 M; lithium salt of methylacetoacetate, 11.7 X lo-’ M; acetoacetyl-CoA, 1.5 X lo-’ M; 3-keto-2-methylbutanoyl-S-thioglycolic acid, 5.4 X lo-’ M; ethyl 3-hydroxy2-methylbutyrate, 6.6 X 10m3M; 3-hydroxy-a-methylbutyryl-CoA, 8 X lo-’ M.
tase activity. The ions examined included the chloride salts of NH:, Cs+, Kf, Li+, M$+, and Ca2+.In addition, EDTA had no effect upon the activity of the enzyme. TABLE
It was reported previously (3) that when the propionyl-CoA condensing enzyme of Ascaris was assayed by coupling the reaction with 2-methylacetoacetyl-CoA reductase, acetyl-CoA inhibited the condensing enzyme or the reductase. In order to clarify this point, the effect of acetylCoA on the reduction of ethyl 2-methylacetoacetate by purified Ascuris reductase was studied (Fig. 4). Indeed, acetyl-CoA inhibited ethyl 2-methylacetoacetate reduction noncompetitively. A 42% inhibition of the reductase activity was observed when both acetyl-CoA and ethyl 2-methylacetoacetate were present in equimolar concentrations (1 X lop3 M). Acetyl-CoA also had the same effect on the crude reductase enzyme activity of the mitochondrial soluble fraction. DISCUSSION
Ascaris was once thought to be unique in its synthesis of the 2-methyl branchedchain volatile acids. However, the important parasites of humans, Paragonimus westermanii (the lung fluke), the saprophytic swamp worm, Alma emini, and the parasitic helminths, Echirwsbma liei and Oesophagostomumradiatum, accumulate 2methylbutyrate (for references, see (17)). Recently, 3-keto-2-methylvalerate and 3hydroxy-2-methylvalerate, both intermediates in the Ascaris pathway, have been identified and are diagnostic in the urine III
KINETIC CONSTANTSOF Ascaris 2-METHYLACETOACETYL-COA
REDUCTASE
vm.u Substrate Ethyl 2-methylacetoacetate Acetoacetyl-CoA 3-Keto-2-methylbutanoyl-S-thioglycolic Ethyl 3-hydroxy-2-methylbutyrate 3-Hydroxy-2-methylbutyryl-CoA NADH NAD+
Km (M)
acid
7 1 2.7 3.3 1.0 6.0 2.7
x x X x x X X
10-s lo-’ lo-’ 10-a 10-d 1O-6 10-s
(I.rmol/min/mg 70.4 25.3 44.6 2.0 5.0 -
protein)
Asuwis 2-METHYLACETOACETYL-CoA
-05
0
0.5
1.0
1.5 l/[S]
REDUCTASE
2.0
91
2.5
(mM)”
FIG. 4. Inhibition of ethyl 2-methylacetoacetate reduction by acetyl-CoA. The reaction mixtures contained in a total volume of 1.0 ml: 0.13 pmol NADH, 100 pmol potassium phosphate buffer, pH 6.0, and 0.86 pg protein. (A) In the absence of acetyl-Cok, (B) in the presence of 2 X 10m3M acetylCoA.
of children suffering from the fatal genetic disease propionic acidemia (18, 19). A propionyl-Cod condensing enzyme and a 2-methylacetoacetyl-CoA reductase activity have been demonstrated in the soluble fraction of the Ascaris mitochondrion (3). These activities are catalyzed by two different proteins. Purified Ascaris 2methylacetoacetyl-CoA reductase lacks propionyl-CoA condensing activity but is presumably important to favor the condensation by pulling off the product, 2methylacetoacetyl-CoA. In addition, purified 2-methylacetoacetyl-CoA reductase was free of enoyl-CoA hydratase activity which, in peroxisomes, copurifies with phydroxyacyl-CoA dehydrogenase activity (13, 14). The molecular weight (M, 64,000) and subunit structure (M* 30,000) reported in
this study for Ascati 2-methylacetoacetylCoA reductase appear to be similar to those assigned to the pig heart and mitochondrial rat liver B-hydroxyacyl-CoA dehydrogenase (15, 16), but different from those assigned to the peroxisomal dehydrogenase. Whereas the mitochondrial enzymes are dimeric proteins comprised of two subunits of the same molecular weight, the peroxisomal enzyme is a monomeric protein with an M, 78,000. In accord with the fact that both 2methylbutyrate and 2-methylvalerate are major end products of Ascaris fermentation, the ethyl esters of 2-methylacetoacetate and 2-methyl-3-keto-valerate were reduced at essentially the same rate by the purified protein. Furthermore, Ascati 2-methylacetoacetyl-CoA reductase was found to be responsible for the NADH-de-
92
SUAREZ
DE MATA
pendent reduction of acetoacetyl-CoA and the NAD+-dependent oxidation of 3-hydroxy-2-methylbutyryl-CoA. A constant activity ratio throughout purification was obtained for acetoacetyl-CoA and ethyl 2methylacetoacetate reductions. In addition, an activity ratio of 2.5 for 3-hydroxy-2methylbutyryl-CoA/ethyl 3-hydroxy-2methylbutyrate oxidations was obtained both with the purified protein and with the enzyme present in the mitochondrial soluble fractions. These findings indicate (i) that the ethyl esters can substitute for the coenzyme A esters (but only when the 2methyl group is present, since no activity was obtained with ethyl acetoacetate), and (ii) Ascaris reductase may act on either linear or branched-chain CoA esters, but has a considerably higher V,,, on the branched-chain CoA esters. In support of the role of the CoA esters as the physiological substrates for the enzyme, the Km value of the purified enzyme for 3-hydroxy2-methylbutyryl-CoA was 33 times lower than that of the corresponding ethyl ester, and the V,,, value was 2.5-fold higher. A comparison of the rates of the reductive versus the oxidative reactions obtained with purified Ascaris reductase with those reported for the corresponding mammalian /3-hydroxyacyl-CoA dehydrogenase from rat liver indicated that the Ascaris enzyme catalyzed the reductive reaction preferentially (V,, reduction/V,,, oxidation = 25). The mammalian enzyme, on the other hand, gave a much lower ratio (3.03), indicating a remarkable difference between the nematode and mammalian enzymes. However, it must be borne in mind that the mammalian enzyme acts physiologically in the oxidative direction for fatty acid breakdown. In contrast, the Ascaris enzyme acts physiologically in the opposite reductive direction for the synthesis of the fatty acids. This also correlates well with the relative substrate specificities of the two enzymes; the former being far more active on the straight-chain acyl-CoA derivatives, while the Ascaris enzyme is considerably more active on the branchedchain esters. The finding that acetyl-CoA noncom-
ET AL.
petitively inhibits the NADH-dependent reduction of ethyl methylacetoacetate by purified enzyme may explain in part the reported inhibition of propionyl-CoA condensing enzyme activity by acetyl-CoA, since the latter enzyme is determined by coupling the products of the condensation reaction with 2-methylacetoacetyl-CoA reductase. A 73% inhibition of propionylCoA condensing enzyme activity was reported when acetyl-CoA and propionylCoA were present in equimolar concentrations (3), whereas a 43% inhibition of the reductase activity of the purified enzyme was obtained when incubated with acetylCoA and ethyl 2-methylacetoacetate under the same assay conditions. In addition, it has been reported (3) that free coenzyme A, which is formed by the acyl-CoA condensation reaction, inhibits the reduction of ethyl 2-methylacetoacetate by the Ascaris mitochondrial soluble fraction. This inhibition, which has been confirmed with the purified enzyme (data not shown), is of the noncompetitive type. These findings suggest that the inhibition of the Ascaris reductase by free coenzyme A may represent a means for controlling the synthesis of the branched-chain fatty acids in this muscle. Similarly, inhibition by acetylCoA may regulate the ratio of 2-methylbutyrate to 2-methylvalerate formed. REFERENCES 1. BUEDING, E., AND YALE, H. (1951) J. Bid Chem 193,441-423. 2. SAZ, H., AND WEIL, A. (1960) J. Bid Chx?m 235, 914-918. 3. SUAREZDE MATA, Z., SAZ, H., AND PASTO, D. (1977) J. Bid Chxm 252,4215-4224. 4. STADTMAN, E. R. (1957) in Methods in Enzymology (Colowick, S. P., and Kaplan, N. O., eds.), Vol. 3, pp. 931-941, Academic Press, New York. 5. VAGELOS, P. R., AND ALBERTS, A. W. (1960) And Bioch 1, 8-16. 6. MCELVAIN, S. M. (1959) J. Amer. Chem Sot. 51, 3124-3130. 7. LIPMANN, F., AND T~TIIE, L. C. (1945) J. Biol Chem 159, 21-28. 8. BRADFORD, M. M. (1976) Ad Biochem 72,248254. 9. LOWRY, 0. H., ROSENBROUGH, N. J., FARR, A. L.,
Asuwk
Z-METHYLACETOACETYL-CoA
AND RANDALL, R. J. (1951) J. Biol
Chem 193,
265-275.
10. SAZ, H. J., AND LESCURE, 0. L. (1969) Camp. Biochxm PhysioL 30,49-60. 11. LAEMMLI, U. K. (1970) Nature (London) 227,680685. 12. DI JESO, F. (1968) J. Bid
Chem. 243, 2022-2023. 13. OSUMI, T., AND HASHIMOTO, T. (1979) B~o&Mz+ Biophys. Res. Commun 89, 580-584. 14. HASHIMOTO, T. (1982) Ann N.Y. Acud sci 386, 5-12. 15. NOYES, B. E., AND BRADSHAW, A. (1973) J. BioL Chem. 248,3052-3059.
REDUCTASE
93
16. OSUMI, T., AND HASHIMOTO, T. (1986) Arch. Biachem Biophys. 203.372-383. 17. SAZ, H. J. (1982) Annu, Rev. PhysioL 43,323-341. 18. MATSUMOTO, I., SHINKA, T., KUHARA, T., OURA, T.,
YAMAMOTO, H., HASE, Y., AOKI, H., ISSHIKI, G., AND TADA, K. (1978) in Recent Developments of Mass Spectrometry in Biochemistry and Medicine (Frigerio, A., ed.), Vol. 1, pp. 203-216, Plenum, New York. 19. TRUSCOTT, R. J. W., PULLIN, C. J., HALPERN, B., HAMMOND, J., HAAN, E., AND DANKS, D. M. (1979) Bknnd Mars Spectrom. 6,294-301.
2-Methylacetoacetyl-Coenzyme Purification ZADILA
SUAREZ
DE
MATA, AND
A Reductase from Ascaris and Properties’
MARIA E. ZARRANZ, HOWARD J. SAZ*
RICHARD
Muscle: LIZARDO,
Departamento de Biologiu Celdar, Universidad Simon Bolivar, Apartah 80659, Camcu.s 1081A, Venezuela, and *Department of Biology, University of Notre Dame, Notre Dame, Indiana 46556 Received January
25, 1983, and in revised form April
29, 1983
2-Methylacetoacetyl-CoA and 3-keto-2-methyl pentanoyl-CoA have been proposed to be intermediates in the synthesis of 2-methylbutyrate and 2-methylvalerate, respectively, by Ascaris lumbricoides muscle. These volatile acids are major fermentation products of Asca& metabolism. 2-Methylacetoacetyl-CoA reductase has been purified 532-fold from Ascaris muscle to yield a homogeneous preparation which contained a single protein species as observed on discontinuous polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulfate. The purification procedure utilized subcellular fractionation, affinity chromatography on NAD+ agarose, and ion-exchange chromatography on DEAE-cellulose. A constant activity ratio for ethyl 2-methylacetoacetate and acetoacetyl-CoA was observed during purification, indicating that the same enzyme catalyzed both reactions. In addition, the purified protein catalyzed the NADH-dependent reduction of ethyl-3-keto-2-methyl pentanoate at essentially the same rate as it did ethyl 2-methylacetoacetate. The purified enzyme is a basic protein with an isoelectric point of 8.45 at 4°C. The molecular weight of the native protein (il4, = 64,000 by exclusion chromatography) and the size of the subunit (ilf, = 30,000 by dodecyl sulfate-polyacrylamide electrophoresis) indicate that the enzyme is composed of two subunits of the same molecular weight. Substrate-specificity studies, undertaken with the purified protein, demonstrated that the ethyl esters can substitute for the coenzyme A derivatives but this substitution results in an active substrate only when a branched 2-methyl group is present. The straight-chain ethyl ester is inactive. Kinetic constants for the substrates and nucleotides were determined. The role of the CoA esters as the physiological substrates for the Ascaris enzyme is substantiated. When assayed in the reductive direction with ethyl 2-methylacetoacetate as substrate, the activity of the purified enzyme was inhibited not only by coenzyme A as previously reported, but also by acetyl-CoA. The physiological implications of these inhibitions are discussed. ucts of carbohydrate metabolism by the parasitic intestinal roundworm, Ascaris lumbricaides var. suum (1). Only traces of n-butyrate, no n-hexanoate, and relatively minor quantities of n-valerate accumulate. This is in accord with the reports that Ascaris, like many other helminths, neither synthesizes long-chain fatty acids de nova nor catabolizes fatty acids appreciably. Isotope studies (2) and more recent enzymological investigations (3) indicate that the branched-chain volatile acids are formed by condensation of acetyl-CoA with
The branched-chain fatty acids, 2-methylbutyrate (2-MB)3 and 2-methylvalerate (2-MV), are the major fermentation prod1 This investigation was supported in part by Grants AI-09483 from the National Institutes of Health, U. S. Public Health Service, and Sl from Consejo National de Investigaciones Cientificas y Tecnologicas (CONICIT), Venezuela. * To whom reprint requests should be sent. a Abbreviations used: 2-M& 2-methylbutyrate; 2MV, 2-methylvalerate; Mes, 4-morpholineethanesulfonic acid. 0003-9861/83 $3.00 Copyright All rights
0 1983 by Academic Press, Inc. of reproduction in any form reserved.
84
Asumi
2-METHYLACETOACETYL-CoA
propionyl-CoA or by condensation of two propionyl-CoA molecules, respectively. The reactions involved are similar to, but in some important respects different from, a reversal of the B-oxidation pathway, particularly in regard to the corresponding enzymes of the host and the parasite. 2-Methylacetoacetyl-CoA, formed by the condensation of acetyl-CoA with propionylCoA, is subsequently reduced by 2-methylacetoacetyl-CoA reductase en route to the synthesis of 2-MB. Reduction of the carbony1 compound is NADH dependent and reversible. The reductive activity was localized in the soluble fraction of the Ascaris mitochondrion, and this activity has been employed to assay for the first reaction of the pathway, i.e., the condensation of the two acyl CoA units (3). The Ascaris enzyme is unique in that the apparent coenzyme A requirement can be substituted for by the ethyl esters of the substrates. However, the ethyl esters are active only if they contain a 2-methyl group. Although activity was reported with acetoacetyl-CoA when the soluble fraction of the Ascaris mitochondrion was the enzyme source (3), the possibility existed that this activity may be due to a different contaminating protein. It is of particular interest, however, that the pig heart L-3hydroxyacyl-CoA dehydrogenase differed from the Ascaris enzyme in that it was specific for the straight-chain CoA esters and no appreciable activity was observed with the branched-chain acyl CoA esters (3). Therefore, Ascaris enzyme serves physiologically almost exclusively for the reduction of the branched-chain compounds. The protein responsible for the reversible reduction of ethyl 2-methylacetoacetate from Ascaris mitochondria now has been purified and characterized. The purified enzyme is a basic protein comprised of two subunits of identical molecular weights. Results demonstrate that the same enzyme is responsible for the NADHdependent reduction of ethyl 3-keto2-methylbutyrate (ethyl methylacetoacetate), ethyl 3-keto-2-methylvalerate, acetoacetyl-CoA, and 3-keto-2-methylbutanoyl-S-thioglycolic acid. The enzyme also
85
REDUCTASE
catalyzed the reverse NAD+ dependent oxidation of ethyl 3-hydroxy-2-methylbutyrate, ethyl 3-hydroxy-2-methylvalerate, and 3-hydroxy-2-methylbutyryl-CoA. It is of particular interest that the Ascaris enzyme catalyzed the reduction reaction more rapidly, since the reduction would be required physiologically for the synthesis of 2-MB and 2-MV. MATERIALS
AND
METHODS
3-Hydroxy-2-methylbutyryl-CoA was synthesized from the mixed anhydride of (dl) 3-hydroxy-2-methylbutyric acid and ethyl chlorocarbonate (4). The mixed anhydride formed was assayed according to the procedure of Lipmann and Tuttle (7). Analysis of the acyl-CoA product by means of high-pressure liquid chromatography indicated that two main peaks were present in a 60~40 ratio. These comprised the two expected pairs of diastereoisomers (3). For the HPLC analysis, an aliquot of the chemically synthesized product was absorbed on a washed Sep-Pak Cl8 cartridge (Waters Associates, Inc. Milford, Mass.). Compounds of high polarity were removed by elution with water, and the acyl-CoA esters were eluted with 50% methanol. Aliquots containing the acyl-CoA ester were applied to a lo-pm Bondapak Cis column (4 mm X 30 cm). Separations were accomplished by elution with 43% methanol in water containing Pit A reagent (Waters Associates). 3-Keto-2-methylbutanoyl-S-thioglycolic acid was synthesized from ethyl 2-methylacetoacetate (Aldrich Chemical Co., Milwaukee, Wise.) by following the procedure described by Vagelos and Alberts (5) for the synthesis of 3-keto-octanoyl-9thioglycolic acid. Ethyl 3-keto-2-methylpentanoate (ethyl 3-keto-2methylvalerate) was synthesized by the condensation of ethyl propionate in the presence of sodium ethoxide (6). Ethyl 3-hydroxy-2-methylbutyrate was obtained by the reduction of ethyl methylacetoacetate (Aldrich) with potassium borohydride (3). Coenzyme A, acetyl-CoA, propionyl-CoA, acetoacetyl-CoA, and NAD+-hexane agarose (AGNAD, type 1) were purchased from P-L Biochemicals (Milwaukee, Wise.), and DEAE-cellulose (DE-52) was purchased from Whatman (Clifton, N. J.). Sephadex G-150, protein standards for molecular-weight determination, polybuffer exchange PBE 94, and polybuffer 96 were Pharmacia (Piscataway, N. J.) products. C,,-agarose employed for hydrophobic chromatography was purchased from Miles Laboratories (Elkhart, Ind.) Acyl-CoA esters were assayed according to Lipmann and Tuttle (7). Protein concentrations were determined by the Bradford method (8) or by the phenol reagent procedure (9) on samples treated with 1% (W/V) sodium dodecyl sulfate. Crystalline bovine
86
SUAREZ
DE MATA
serum albumin was used as standard with both methods. Mitochondria were prepared from dissected Ascaris muscle strips in 0.2 M sucrose, 5 mM EDTA, and 0.15% crystalline bovine serum albumin, pH 7.4, as described previously (10). 2-Methylacetoacetyl-CoA reductase was assayed spectrophotometrically at 340 nm by means of a modification of the method of Suarez de Mata et aL (3). The potassium phosphate buffer, pH 6.0, was replaced by 75 mM Mes, pH 5.5. Unless otherwise stated, the substrate employed for assay was ethyl 2-methylacetoacetate. Activity in the reverse (oxidative) direction was measured spectrophotometrically at 340 nm by following the concomitant reduction of NAD+ as described by Suarez de Mata et al (3). One unit of enzyme activity is equivalent to one micromole of substrate transformed per minute. Electrophoresis of protein was carried out on polyacrylamide slab gels in the presence of 0.1% sodium dodecyl sulfate with the discontinuous buffer system and gel formulation of Laemmli (11). Samples were denatured prior to electrophoresis by heating at 100°C for 30 min in 1% sodium dodecyl sulfate containing 1% mercaptoethanol.
Pur$ication of .%Methylacetoacetyl-CoA Reductase 1. Isolation of mitochundrial soluble fraction. 2Methylacetoacetyl-CoA reductase activity is localized and was isolated from the soluble fraction of the Ascoris mitochondrion (3). Washed mitochondria obtained from 60 g (wet wt) of adult female Ascaris muscle were resuspended to 27 ml with 0.02 M potassium phosphate buffer, pH 6.0. This suspension was freeze-thawed and subsequently sonicated in the cold with 10 pulses of 30 s each with 60-s cooling intervals in a Biosonic III sonicator, fitted with a microtip at a power setting of 30 W (Bronwill Scientific, Rochester, N. Y.) The mitochondrial soluble fraction was obtained after centrifugation at 269,000g for 30 min. 2. Ammonium sulfate precipitation Sufficient solid ammonium sulfate was added to the mitochondrial soluble fraction to make the solution 80% saturated (51.6 g/200 ml) (12). After standing for 30 min at 4”C, the mixture was centrifuged at 13,500g for 30 min. The yellow precipitate obtained was made to 3 ml with 0.02 M potassium phosphate buffer, pH 6.0. The sample then was desalted by passage over a column (1.5 x 5 cm) of Sephadex G-50 equilibrated with 20 rnM potassium phosphate buffer, pH 6.0. 3. NAD+-Agarose a&nity chromatography. The above sample was applied to an NAD+-agarose column (0.9 X 8 cm) equilibrated with 20 mM potassium phosphate buffer, pH 6.0. After a wash with 20 ml of the equilibration buffer, elution was carried out with 20 ml of a 2.6 mM NADH solution in 20 mM potassium
ET AL.
phosphate buffer, pH 8.0. Fractions of 1.0 ml were collected and the enzymatically active fractions were pooled. 4 DEAE-ceUulcse and hydrqdnbic chromatography. The active fractions eluted from the NAD+-agarose column were applied to a DEAE-cellulose column (0.2 X 21 cm) which was equilibrated with 20 mM potassium phosphate buffer, pH 7.5. Elution of the column was carried out with 20 ml of the equilibration buffer. Only occasionally was a minor contaminant present in this eluate, as indicated by electrophoresis on sodium dodecyl sulfate-acrylamide slab gels. Only when this contaminant was present was the additional step added. This contaminant was eliminated by subjecting the sample to hydrophobic chromatography on a Cl0 column (0.9 X 3.5 cm) equilibrated in the above buffer. The column was washed sequentially with 8 ml each of equilibration buffer containing 0 and 1.0 M NaCl, and eluted with 10 ml of 1.0 M NaCl in equilibration buffer containing 50% ethylene glycol. The sample eluted had a slightly higher specific activity and contained 78% of the initial activity. When necessary for gel chromatography, DEAEcellulose and Cl0 active fractions were concentrated by ultrafiltration using YM-10 Diaflo membranes (Amicon Corp., Danvers, Mass.). The isoelectric point of the Ascaris 2-methylacetoacetyl-CoA reductase was determined by chromatofocusing on a column (0.9 X 30 cm) of polybuffer exchanger PBE 94 equilibrated with 0.025 M ethanolamine-acetic acid buffer, pH 9.2, and eluted with polybuffer 96 adjusted to pH 6.0 with 1.0 M acetic acid.
RESULTS
Enzyme Pur$cation 2-Methylacetoacetyl-CoA reductase was purified from the soluble fraction of the Ascaris mitochondrion by means of affinity chromatography on NAD+-agarose and ion-exchange chromatography on DEAEcellulose. The purified enzyme had a specific activity of 55 units/mg of protein (Table I) after a 538-fold purification at a yield of 24%. The value of 538-fold purification was calculated by taking into consideration that the isolation of the mitochondrial soluble fraction from crude homogenates resulted in a 19-fold purification in terms of 2-methylacetoacetyl-CoA reductase activity. It should be emphasized that at the NAD+-agarose step both propionyl-CoA condensing enzyme and enoyl-CoA hydratase activities are separated from the reductase. These activities, which also are
Ascuri.s
2-METHYLACETOACETYL-CoA TABLE
I
PURIFICATIONOF Ascaris 2-METHYLACETOACETYL-COA 2-Methylacetoacetyl-CoA
Fraction 1. Mitochondrial soluble” 2. Ammonium sulfate (O-80%) 3. NAD-Agarose 4. DEAE-cellulose
87
REDUCTASE
REDUCTASE
reductase activity
Specific activity ratio: Ethyl methylacetoacetate/ Acetoacetyl-CoA
Total protein (md
Total (units)
47.5
92.2
1.9
100
1
2.68
32.6 3.16 0.4
78.4 44.5 22.3
2.4 14.1 55.1
85 48 24
1.2 7.3 28.3
3.2 2.6 3.0
a Isolation of the mitochondrial terms of 2-methylacetoacetyl-CoA
Specific activity (units/mg protein)
Percentage recovery
soluble fraction from crude homogenate reductase activity.
involved in the synthesis of 2-MB and 2MV, did not bind to NAD+-agarose and were recovered in the wash of the column. The composition of the fractions at the various stages of the purification was followed by dodecyl sulfate-polyacrylamide gel electrophoresis (Fig. 1). The final preparations were homogeneous on these gels, a single protein band being observed. This band was present in approximately equal intensity when protein amounts corresponding to equal amounts of reductase activity were applied to the gel and, presumably, would be derived from the active enzyme.
Fold purification
resulted in a Is-fold
purification
in
The isoelectric point of the Ascaris enzyme was determined by chromatofocusing, as described under Materials and Methods. The value obtained by this method was equal to a pH of 8.45. This pH value was consistent with the observed behavior of the enzyme on DEAE-cellulose. This technique also served as an additional criterion for homogeneity, since when the purified preparation was first applied to the same column, but equilibrated with 0.025 M imidazole HCl buffer, pH 7.4, and eluted with polybuffer 74 set to pH 4.0 (data not shown), only one symmetrical protein peak which overlapped with the activity peak was observed.
Chamcterization of LMethylacetoacetyG CoA Reductase.
Substrate Specificity of the Reductase
The molecular weight of the purified Ascaris 2-methylacetoacetyl-CoA reductase was determined chromatographically on a column (1 X 43 cm) of Sephadex G-150, eluted with 10 mM potassium phosphate buffer, pH 7.5. The enzyme eluted at a position corresponding to M, 64,000.The same results were obtained employing an AcA 34 Ultrogel column (1 X 40 cm) equilibrated with 20 mM potassium phosphate buffer, pH 7.5. The number and size of the subunit(s) in the native protein were investigated by means of sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The dissociated enzyme formed a single protein band with an electrophoretic mobility corresponding to M, 30,000.
The HPLC spectrum of the synthetic 3-hydroxy-2-methylbutyryl-CoA showed two major peaks in a 60:40 ratio. These peaks comprised the two expected pairs of diastereoisomers. Initial rates of oxidation of 3-hydroxy-2-methylbutyryl-CoA and the reduction of 3-keto-2-methylbutanoyl-Sthioglycolate, respectively, were determined at increasing concentrations of each substrate (Figs. 2 and 3). Normal Michaelis-Menten responses were observed with these synthetic compounds. Comparative substrate-specificity studies were examined (Table II). The ethyl esters of both 2methylacetoacetate and 3-keto-2-methylpentanoate, the presumed precursors of 2MB and 2-MV, were reduced at essentially the same rate by the purified enzyme. No
88
SUAREZ
DE MATA
ET AL.
when a branched methyl group is present since no activity could be detected on ethyl acetoacetate, whereas the CoA ester of acetoacetate did show activity. The chemically synthesized thioglycolate ester of 3keto-2-methylbutyrate was reduced by the purified enzyme at a rate approaching that obtained with the ethyl ester. The reductase reaction was reversible, but the ethyl esters of both 3-hydroxy-2methylbutyrate and 3-hydroxy-2-methylvalerate were oxidized at approximately one-twentieth the rate of the reverse, reduction reaction. The difference between the rate of the two reactions became somewhat less when the coenzyme A ester of 3-hydroxy-2-methylbutyrate was employed as substrate for the oxidative reaction, but remained approximately one-tenth that of the reduction reaction. This is of particular interest, since physiologically the enzyme would function primarily in the faster, reductive direction. The purified reductase was specific for NAD+. Neither NADP+ nor NADPH was utilized. 1
234567
8
9
10
FIG. 1. Purification of 2-methylacetacetyl-CoA reductase by discontinuous gel electrophoresis in 0.1% sodium dodecyl sulfate. A 9% acrylamide running gel with 3% stacking gel was formulated and run as described under Materials and Methods. The contents of each slot were 1, reference proteins; 2, mitochondrial soluble fraction; 3, ammonium sulfate pellet; 4, NAD-agarose fraction; 5, DEAE-cellulose fraction; 6,7, and 8, CIO-hydrophobic fraction; 9, reference proteins; 10, chymotrypsinogen. To slots 2 through 6 was added an aliquot from the respective purification step containing 0.025 units of reductase activity. Slots 7 and 8 contained 0.13 and 0.08 units, respectively.
activity was observed with the ethyl ester of acetoacetate or with the lithium salt of methylacetoacetate, confirming the requirement for both the branched methyl and ester groups. Acetoacetyl-CoA was reduced by the purified enzyme, but only at approximately one-third the rate of the ethyl esters of the branched-chain acids. In addition, a constant activity ratio of ethyl methylacetoacetate/acetoacetyl-CoA reduction was observed during purification (Table I). It appears that the ethyl ester can substitute for the CoA derivative only
Kinetic Constants of the Ascaris Reductase The Km values obtained with Ascaris reductase were approximately one order of magnitude higher when the ethyl esters were employed as substrates, compared with the CoA derivatives (Table III). Most important, the Km for 3-hydroxy-2-methylbutyryl-CoA was 33 times lower than that for the corresponding ethyl ester and compared more favorably with the values obtained for the physiological nucleotides, NADH and NAD+. The V,,, values obtained for both the ethyl and thioglycolate esters of 2-methylacetoacetate were approximately the same, and three times higher than the V,,, obtained for acetoacetyl-CoA. In addition, more than a twofold increase in the I’,,,, was observed with the CoA ester of 3-hydroxy-2-methylbutyrate in comparison to the corresponding ethyl ester. Neither monovalent nor divalent cations had an appreciable effect upon the purified Ascaris ethyl 2-methylacetoacetate reduc-
Asmti
2-METHYLACETOACETYL-CA
89
REDUCTASE
0.7
0.6
0.5
f \ a
0.4
0
0.3
0.2
0.1
1
0
02 [b-Hydroxy-2-
I
0.4
L
0.6
methylbutyryl
0.6
t
IO
-I 1.;2
COA] mM.
FIG. 2. Dependence of the initial rate of 3-hydroxy-2-methylbutyryl-CoA oxidation on substrate concentration. Assay conditions were as described under Materials and Methods. Assays contained 2.8 fig of protein. Substrate concentrations were determined by the method of Lipmann and Tuttle (7). I
I
6 /
[J-K&o
-2-mathylbuionoyl-S-
thioglycolic
acid ] mht
FIG. 3. Dependence of the initial rate of the reduction of 3-keto-2-methylbutanoyl-S-thioglycolic acid on substrate concentration. Assay conditions were as described under Materials and Methods. Assay contained 3.4 pg of protein. Substrate concentrations were determined by the method of Lipmann and Tuttle (7).
90
SUAREZ TABLE
DE MATA
Eflect of Acetyl-CoA on b Methylacetoacetyl-CoA Reductase Activity
II
SUBSTRATE SPECIFICITY OF PURIFIED Ascaris 2-METHYLACETOACETYL-CoA REDUCTASE Activity” (pmol/min/ mg protein)
Substrate Propionyl-CoA Ethyl 2-methylacetoacetats Ethyl 3-keto-2-methylpentanoate Ethyl Acetoacetate Lithium salt of methylacetoacetate Acetoacetyl-CoA 3-Keto-2-methylbutanoyl-Sthioglycolic acid Ethyl 3-hydroxy-2-methylvalerate + NAD+ Ethyl 3-hydroxy-2-methylbutyrate + NAD+ 3-Hydroxy-2-methylbutyryl-CoA + NAD+
ET AL.
0 49.1 51.0 0 0 16.2 37.0 3.4 2.0 5.0
a Enzyme activities were determined spectrophotometrically as described under Materials and Methods. The molar concentrations of the substrates employed were propionyl-CoA, 2 X 10e3 M; ethyl 2-methylacetoacetate, 11.7 X 10-s M; ethyl 3-keto-2-methylpentanoate, 11.7 X 10-s M; ethyl acetoacetate, 11 X 10e3 M; lithium salt of methylacetoacetate, 11.7 X lo-’ M; acetoacetyl-CoA, 1.5 X lo-’ M; 3-keto-2-methylbutanoyl-S-thioglycolic acid, 5.4 X lo-’ M; ethyl 3-hydroxy2-methylbutyrate, 6.6 X 10m3M; 3-hydroxy-a-methylbutyryl-CoA, 8 X lo-’ M.
tase activity. The ions examined included the chloride salts of NH:, Cs+, Kf, Li+, M$+, and Ca2+.In addition, EDTA had no effect upon the activity of the enzyme. TABLE
It was reported previously (3) that when the propionyl-CoA condensing enzyme of Ascaris was assayed by coupling the reaction with 2-methylacetoacetyl-CoA reductase, acetyl-CoA inhibited the condensing enzyme or the reductase. In order to clarify this point, the effect of acetylCoA on the reduction of ethyl 2-methylacetoacetate by purified Ascuris reductase was studied (Fig. 4). Indeed, acetyl-CoA inhibited ethyl 2-methylacetoacetate reduction noncompetitively. A 42% inhibition of the reductase activity was observed when both acetyl-CoA and ethyl 2-methylacetoacetate were present in equimolar concentrations (1 X lop3 M). Acetyl-CoA also had the same effect on the crude reductase enzyme activity of the mitochondrial soluble fraction. DISCUSSION
Ascaris was once thought to be unique in its synthesis of the 2-methyl branchedchain volatile acids. However, the important parasites of humans, Paragonimus westermanii (the lung fluke), the saprophytic swamp worm, Alma emini, and the parasitic helminths, Echirwsbma liei and Oesophagostomumradiatum, accumulate 2methylbutyrate (for references, see (17)). Recently, 3-keto-2-methylvalerate and 3hydroxy-2-methylvalerate, both intermediates in the Ascaris pathway, have been identified and are diagnostic in the urine III
KINETIC CONSTANTSOF Ascaris 2-METHYLACETOACETYL-COA
REDUCTASE
vm.u Substrate Ethyl 2-methylacetoacetate Acetoacetyl-CoA 3-Keto-2-methylbutanoyl-S-thioglycolic Ethyl 3-hydroxy-2-methylbutyrate 3-Hydroxy-2-methylbutyryl-CoA NADH NAD+
Km (M)
acid
7 1 2.7 3.3 1.0 6.0 2.7
x x X x x X X
10-s lo-’ lo-’ 10-a 10-d 1O-6 10-s
(I.rmol/min/mg 70.4 25.3 44.6 2.0 5.0 -
protein)
Asuwis 2-METHYLACETOACETYL-CoA
-05
0
0.5
1.0
1.5 l/[S]
REDUCTASE
2.0
91
2.5
(mM)”
FIG. 4. Inhibition of ethyl 2-methylacetoacetate reduction by acetyl-CoA. The reaction mixtures contained in a total volume of 1.0 ml: 0.13 pmol NADH, 100 pmol potassium phosphate buffer, pH 6.0, and 0.86 pg protein. (A) In the absence of acetyl-Cok, (B) in the presence of 2 X 10m3M acetylCoA.
of children suffering from the fatal genetic disease propionic acidemia (18, 19). A propionyl-Cod condensing enzyme and a 2-methylacetoacetyl-CoA reductase activity have been demonstrated in the soluble fraction of the Ascaris mitochondrion (3). These activities are catalyzed by two different proteins. Purified Ascaris 2methylacetoacetyl-CoA reductase lacks propionyl-CoA condensing activity but is presumably important to favor the condensation by pulling off the product, 2methylacetoacetyl-CoA. In addition, purified 2-methylacetoacetyl-CoA reductase was free of enoyl-CoA hydratase activity which, in peroxisomes, copurifies with phydroxyacyl-CoA dehydrogenase activity (13, 14). The molecular weight (M, 64,000) and subunit structure (M* 30,000) reported in
this study for Ascati 2-methylacetoacetylCoA reductase appear to be similar to those assigned to the pig heart and mitochondrial rat liver B-hydroxyacyl-CoA dehydrogenase (15, 16), but different from those assigned to the peroxisomal dehydrogenase. Whereas the mitochondrial enzymes are dimeric proteins comprised of two subunits of the same molecular weight, the peroxisomal enzyme is a monomeric protein with an M, 78,000. In accord with the fact that both 2methylbutyrate and 2-methylvalerate are major end products of Ascaris fermentation, the ethyl esters of 2-methylacetoacetate and 2-methyl-3-keto-valerate were reduced at essentially the same rate by the purified protein. Furthermore, Ascati 2-methylacetoacetyl-CoA reductase was found to be responsible for the NADH-de-
92
SUAREZ
DE MATA
pendent reduction of acetoacetyl-CoA and the NAD+-dependent oxidation of 3-hydroxy-2-methylbutyryl-CoA. A constant activity ratio throughout purification was obtained for acetoacetyl-CoA and ethyl 2methylacetoacetate reductions. In addition, an activity ratio of 2.5 for 3-hydroxy-2methylbutyryl-CoA/ethyl 3-hydroxy-2methylbutyrate oxidations was obtained both with the purified protein and with the enzyme present in the mitochondrial soluble fractions. These findings indicate (i) that the ethyl esters can substitute for the coenzyme A esters (but only when the 2methyl group is present, since no activity was obtained with ethyl acetoacetate), and (ii) Ascaris reductase may act on either linear or branched-chain CoA esters, but has a considerably higher V,,, on the branched-chain CoA esters. In support of the role of the CoA esters as the physiological substrates for the enzyme, the Km value of the purified enzyme for 3-hydroxy2-methylbutyryl-CoA was 33 times lower than that of the corresponding ethyl ester, and the V,,, value was 2.5-fold higher. A comparison of the rates of the reductive versus the oxidative reactions obtained with purified Ascaris reductase with those reported for the corresponding mammalian /3-hydroxyacyl-CoA dehydrogenase from rat liver indicated that the Ascaris enzyme catalyzed the reductive reaction preferentially (V,, reduction/V,,, oxidation = 25). The mammalian enzyme, on the other hand, gave a much lower ratio (3.03), indicating a remarkable difference between the nematode and mammalian enzymes. However, it must be borne in mind that the mammalian enzyme acts physiologically in the oxidative direction for fatty acid breakdown. In contrast, the Ascaris enzyme acts physiologically in the opposite reductive direction for the synthesis of the fatty acids. This also correlates well with the relative substrate specificities of the two enzymes; the former being far more active on the straight-chain acyl-CoA derivatives, while the Ascaris enzyme is considerably more active on the branchedchain esters. The finding that acetyl-CoA noncom-
ET AL.
petitively inhibits the NADH-dependent reduction of ethyl methylacetoacetate by purified enzyme may explain in part the reported inhibition of propionyl-CoA condensing enzyme activity by acetyl-CoA, since the latter enzyme is determined by coupling the products of the condensation reaction with 2-methylacetoacetyl-CoA reductase. A 73% inhibition of propionylCoA condensing enzyme activity was reported when acetyl-CoA and propionylCoA were present in equimolar concentrations (3), whereas a 43% inhibition of the reductase activity of the purified enzyme was obtained when incubated with acetylCoA and ethyl 2-methylacetoacetate under the same assay conditions. In addition, it has been reported (3) that free coenzyme A, which is formed by the acyl-CoA condensation reaction, inhibits the reduction of ethyl 2-methylacetoacetate by the Ascaris mitochondrial soluble fraction. This inhibition, which has been confirmed with the purified enzyme (data not shown), is of the noncompetitive type. These findings suggest that the inhibition of the Ascaris reductase by free coenzyme A may represent a means for controlling the synthesis of the branched-chain fatty acids in this muscle. Similarly, inhibition by acetylCoA may regulate the ratio of 2-methylbutyrate to 2-methylvalerate formed. REFERENCES 1. BUEDING, E., AND YALE, H. (1951) J. Bid Chem 193,441-423. 2. SAZ, H., AND WEIL, A. (1960) J. Bid Chx?m 235, 914-918. 3. SUAREZDE MATA, Z., SAZ, H., AND PASTO, D. (1977) J. Bid Chxm 252,4215-4224. 4. STADTMAN, E. R. (1957) in Methods in Enzymology (Colowick, S. P., and Kaplan, N. O., eds.), Vol. 3, pp. 931-941, Academic Press, New York. 5. VAGELOS, P. R., AND ALBERTS, A. W. (1960) And Bioch 1, 8-16. 6. MCELVAIN, S. M. (1959) J. Amer. Chem Sot. 51, 3124-3130. 7. LIPMANN, F., AND T~TIIE, L. C. (1945) J. Biol Chem 159, 21-28. 8. BRADFORD, M. M. (1976) Ad Biochem 72,248254. 9. LOWRY, 0. H., ROSENBROUGH, N. J., FARR, A. L.,
Asuwk
Z-METHYLACETOACETYL-CoA
AND RANDALL, R. J. (1951) J. Biol
Chem 193,
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