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Clofibric acid, phenylpyruvate, and dichloroacetate inhibition of branched-chain α-ketoacid dehydrogenase kinase in vitro and in perfused rat heart
ARCHIVES
OF BIOCHEMISTRY
Vol. 231, No. 1, May
AND BIOPHYSICS 15, pp. 58-66, 1984
Clofibric Acid, Phenylpyruvate, and Dichloroacetate Branched-Chain cw-Ketoacid Dehydrogenase in Vitro and in Perfused Rat Heart’ RALPH of Bicdemistry,
Department
Received
PAXTON Indiana
September
AND
University
ROBERT School
‘7, 1983, and in revised
Inhibition of Kinase
A. HARRIS’
of Medicine, form
Indianapolis,
January
Indiana
46223
23, 1984
Branched-chain a-ketoacid dehydrogenase kinase, purified from rabbit liver, was inhibited by clofibric acid, phenylpyruvate, and dichloroacetate in a mixed manner relative to ATP. 14,,values relative to 75 ELM ATP were 0.33,1.7, and 3.0 mM, respectively. Inhibition of the kinase by acetate, pyruvate, and lactate was minimal; whereas a phydroxyphenyl substitution of these compounds increased their potency as kinase inhibitors, a phenyl substitution gave the most potent inhibitors. Clofibric acid, phenylpyruvate, and dichloroacetate activated branched-chain a-ketoacid dehydrogenase in perfused rat hearts. Perfusate concentrations that gave 50% activation (A& were 0.1, 0.32, and 0.63 mM, respectively. A50 concentrations of clofibric acid and phenylpyruvate also increased flux (decarboxylation of a-keto[l-‘4C]isovalerate) through branched-chain cu-ketoacid dehydrogenase in perfused rat heart. These findings suggest that, although clofibric acid and phenylpyruvate can inhibit substrate utilization by the branchedchain cu-ketoacid dehydrogenase complex, the major effect of these compounds on branched-chain amino acid metabolism is due to inhibition of branched-chain a-ketoacid dehydrogenase kinase with subsequent activation of and increased flux through the complex.
Branched-chain a-ketoacid dehydrogenase complex (EC 1.2.4.4) is a mitochondrial multienzyme complex that catalyzes the oxidative decarboxylation of cy-ketoisocaproate, a-ketoisovalerate, and a-ketofi-methylvalerate (transamination products of Leu, Val, and Ile, respectively). The complex is rate limiting for the metabolism of branched-chain amino acids, except in liver where transamination is rate limiting (1). Activity of the complex is regulated
competitively by product inhibition (2) and covalently through phosphorylation, which decreases V,,, (3-5). The phosphorylation state of the complex will be determined by the combined actions of a kinase, which has been partially characterized (3), and by a phosphatase that has not yet been isolated or characterized. Thus, branchedchain amino acid metabolism will be determined by the flux through the complex which is regulated at two levels, with the maximum flux (i.e., V,,,) dependent upon the phosphorylation state (i.e., relative kinase to phosphatase activity) and modulation of this maximum flux by substrate and product influences. Alteration of phosphorylation state through kinase inhibition has been shown for two mitochondrial multienzyme complexes (i.e., branched-chain Lu-ketoacid de-
i This work was supported in part by NIH Research Grants AM19259 and 5 SO7 RR5371, and the Grace M. Showalter Residuary Trust. A preliminary report of this work was presented at the 1983 meeting of the American Society of Biological Chemists in San Francisco, Calif. ‘To whom correspondence and reprint requests should be addressed. 0003-9861/84 Copyright All rights
$3.00
0 1984 by Academic Press, Inc. of reproduction in any form reserved.
58
INHIBITORS
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BRANCHED-CHAIN
(Y-KETOACID
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59
hydrogenase and pyruvate dehydrogenase, concentration of these compounds needed EC 1.2.4.1) to decrease the phosphorylation for these effects in perfused rat heart is state (increased percentage active form) similar to therapeutic or physiological concentrations, the effects of these comand to increase the flux through these pounds on branched-chain amino acids complexes. cr-Chloroisocaproate inhibits isolated branched-chain ar-ketoacid dehymay be mediated through activation of the complex. drogenase kinase, and decreases the phosphorylation state of and increases the flux through the complex in perfused rat heart EXPERIMENTAL PROCEDURES (6, 7). Similarly, dichloroacetate inhibits isolated pyruvate dehydrogenase kinase, Materials. Reagents were obtained as previously and decreases the phosphorylation state of given (3, 6, 17), with the following exceptions: aand increases the flux through this complex Kewl-“Cjisovalerate was from Amersham Corp.; and in perfused rat heart and other tissues (8, dichloroacetic acid and Scinti Verse II were from 9). Thus, alterations in phosphorylation Fisher Scientific Company. cr-Chloroisocaproate was a gift from Dr. Robert J. Strohscheim and Dr. Ronald state of these complexes, which influences Simpson of Sandoz, Inc. V max9 appears to determine maximum flux Branched-chain &Aoacid dehydrogenase isolatiolz under a given set of conditions. and assay. Rabbit liver branched-chain cy-ketoacid Several compounds of medical or biodehydrogenase with intrinsic kinase activity was isochemical interest affect branched-chain lated and assayed as given in (17). The spectrophoamino acid metabolism and may therefore tometric assay of the complex was done at 30°C as influence the phosphorylation state of previously described (3), with other conditions as given branched-chain a-ketoacid dehydrogenase. in the text. Clofibric acid (2-(4-chlorophenoxy)-2Branched-chain a-ketoacid dehydrogenase kinase methylpropionic acid), the active agent of assay. Branched-chain a-ketoacid dehydrogenase ki32P as previously clofibrate (ethyl ester of clofibric acid), is nase was assayed by protein-bound described (17). an antihyperlipidemic and antihyperchoIsolation of broa&specifcity protein phosphutase. lesterolemic agent that also reduces blood Broad-specificity protein phosphatase isolation and branched-chain amino acids concentration assay was as previously described (17). in humans (10) and rats (11). PhenylkeHeart pmfusia studies. Hearts from 300- to 500tonuria, an inherited metabolic disorder g male Wistar rats were perfused by the flow-through involving phenylalanine metabolism, leads Langendorff procedure as previously described (17). to appearance of phenylpyruvate in the Hearts were perfused at 37°C for the indicated time blood (12) and reduced concentration of with Krebs-Henseleit buffer containing 10 mM glublood branched-chain amino acids (13). cose, 20 mu/ml bovine insulin, 0.1 mM cY-ketoisovalSimilarly, a large dose of phenylalanine erate (which had a specific activity of 30 to 50 dpm/ nmol of cu-keto[l-%]isovalerate for branched-chain administered to rats leads to reduced concu-ketoacid dehydrogenase flux measurement), and centrations of blood and liver branchedadditions as given in the test. chain amino acids (14). Dichloroacetate inDetermination of percentage active from branchedhibits isolated rabbit liver branched-chain chain cu-ketoacid dehydrogenase in perfused rat heart. cy-ketoacid dehydrogenase kinase (3) and of the percentage active form of increases flux through branched-chain (Y- The determination the complex was as previously given (17). ketoacid dehydrogenase in perfused rat Branched-chain ol-ketoacid dehydrogenase j&x hearts (15,16). However, the sensitivity of measurements in perfused rat heart. Hearts were perthe complex to activation by dichloroacefused as above (with a-keto[l-‘“Clisovalerate) for 15 tate has not been determined in perfused min and then additions to perfusate were made as rat heart. given. At 40 min from the start of perfusion the perThe results of this study demonstrate fusate from the heart was collected for 1 min in a that clofibric acid and phenylpyruvate can 25-ml Erlenmeyer flask that contained 0.5 mmol KOH inhibit isolated branched-chain a-ketoacid and 50 pmol EDTA. The collected perfusate volume dehydrogenase kinase, activate the comwas determined gravimetrically and the flask was plex, and increase the flux through the fitted with a serum cap containing a plastic center well. The collected perfusate was then acidified with complex in perfused rat heart. Since the
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0.3 ml of 0.8 M KHzPOl and 3.5 M H,PO,. Liberated COP was collected in the center well which contained 0.3 ml of 1 M KOH. After 1.5 to 2 h in a shaking water bath at 20°C the center wells were removed and placed in ScintiVerse II, and radioactivity was determined. Kinetic analyses were performed as previously given (3). All statistical tests and associated tables were from Ref. (18).
RESULTS
Inhibition of branched-chain cu-ketoacid dehydrogenase kinase by cloJibric acid, phenylpymvate, and dichloroacetate. Clofibric acid (Fig. lA), phenylpyruvate (Fig. lB), and dichloroacetate (data not shown) showed mixed inhibition of branched-chain a-ketoacid dehydrogenase kinase relative to ATP. The exact mechanism of inhibition was not clear since the reciprocal plots of velocity versus ATP concentrations did not have a common intersection and the replots of the y intercepts versus inhibitor concentration were apparently nonlinear. Since the mechanism of inhibition was not known the quantitation of the relative potency of these compounds to inhibit the kinase could not be determined from these data. However, readily comparable values of inhibitor potency were determined with a single ATP concentration (75 PM). IbOvalues (amount of compound needed to inhibit 40% of total branched-chain a-ketoacid dehydrogenase kinase activity; Fig. 2) were selected as the comparison value since the inhibition was nonlinear and it appeared that the inhibition pattern at 40% was fairly uniform between the different compounds. The inhibitor concentrations needed to achieve 40% inhibition (IU value) of the total branched-chain a-ketoacid dehydrogenase kinase activity were 0.33,1.7, and 3.0 mM for clofibric acid, phenylpyruvate, and dichloroacetate, respectively (Fig. 2). It should be noted that these estimates of the relative potency of these compounds to inhibit the kinase, although not independent of ATP concentration, were found to correspond to the relative abilities of these compounds to activate the complex in perfused rat heart as shown below.
FIG. 1. Inhibition of branched-chain cY-ketoacid dehydrogenase kinase by clofibric acid (A) and phenylpyruvate (B). Branched-chain cY-ketoacid dehydrogenase kinase was assayed under the conditions described under Experimental Procedures, with various concentrations of either clofibric acid (A) or phenylpyruvate (B) and ATP as indicated in the figure. Velocity is nmol 9 bound 20 min? mg protein-‘.
Since elevated phenylpyruvate levels associated with phenylketonuria are also associated with elevated levels of other abnormal metabolites of phenylpyruvate (e.g. phenylacetate and phenyllactate (19)) several other compounds associated with phenylketonuria were also tested as inhibitors of branched-chain cu-ketoacid dehydrogenase kinase (Table I). While acetate, pyruvate, and lactate had minimal inhibitory effects, addition of a phenyl
INHIBITORS
OF
BRANCHED-CHAIN
a-KETOACID
FIG. 2. Inhibition (I, values) of branched-chain cY-ketoacid dehydrogenase kinase by dichloroacetate (A), phenylpyruvate (B), and clofibric acid (C). Branched-chain a-ketoacid dehydrogenase kinase was assayed as in Fig. 1, except assay time was 30 min and ATP concentration was 75 PM with other additions as given in the figure. Under these conditions 100% branched-chain cY-ketoacid dehydrogenase kinase activity (nmol 32p bound 30 min-’ mg protein-‘) was 5.13 f 0.13 (it + SE) for 33 determinations.
DEHYDROGENASE
and 4.3% f 1.0 (12) in the presence of (Yketoisovalerate. The total activity (nmol min-’ g wet wt-’ at 30°C with 1 mM (Yketoisovalerate) for 84 hearts done in a similar manner (combined number of hearts in this study and another (17)) was 248 f 40 (ii f SD). Since under these conditions the complex must be almost completely phosphorylatecl, any compound that selectively inhibits branched chain cz-ketoacid dehyclrogenase kinase should result in an elevated percentage active form of and flux through the complex. The percentage active form of branchedchain a-ketoacid dehydrogenase in perfused rat heart was increased with increasing concentrations of clofibric acid, phenylpyruvate, and dichloroacetate (Fig. 3). The concentration of clofibric acid, phenylpyruvate, and dichloroacetate needed to achieve 50% activation (Am value) was 0.1,0.32, and 0.63 mM, respectively. It cannot be excluded that these compounds activated the complex by stimulation of the branched-chain a-ketoacid dehydrogenase phosphatase. However, the fact that these compounds inhibited the isolated branchedchain a-ketoacid dehydrogenase kinase and that the relative inhibitory abilities of these compounds to inhibit the isolated kinase are similar to their abilities to actiTABLE
group greatly increased potency of these carboxylic acids to inhibit branched-chain a-ketoacicl dehydrogenase kinase. However, a p-hydroxy substitution on the phenyl group reduced the potency of these compounds. Phenylalanine (2 mM) did not inhibit the kinase (data not shown). Activation of branched-chain a-h&x&d dehydrogenase in perfused rat heart by clujibric acid, phen&yruvate, and dichbm acetate. Rat hearts perfused with glucose have a very low activity of and flux through branched-chain a-ketoacid dehydrogenase (this study and (15, 16, 20, 21)). The presence of 0.1 mM cY-ketoisovalerate did not change the percentage active form of branched-chain a-ketoacid dehydrogenase in perfused rat hearts with ii + SE [number of hearts] of 3.1% + 0.6 (3) in the absence
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I
EFFECTS OF VARIOUS COMPOUNDS ON INHIBITION BRANCHED-CHAIN PKETOACID DEHYDROGENASE’
Compound
Percentage inhibition
(2 mM)
Acetate Phenylacetate o-Hydroxyphenylacetate pHydroxyphenylacetate
15 32 6 45
Pyruvate Phenylpyruvate p-hydroxyphenylpyruvate
8 50 30
Lactate Phenyllactate pHydroxyphenyllactate
0 24 12
“Determined as given centration was 0.2 mM.
OF
in Fig.
2 except
ATP
con-
PAXTON
AND
HARRIS
3.3), while phenylpyruvate was competitive (Fig. 4B) with an apparent Ki of 0.05 mM. No qualitative difference in the type of inhibition has been seen when different branched-chain a-ketoacids were used to analyze cY-chloroisocaproate inhibition (6), and therefore the use of any LYketoacid substrate appears to adequately describe the type of inhibition. Ir,,, values (50% inhibition of total activity) with 56 PM a-ketoisovalerate for clofibric acid and phenylpyruvate were 0.08 and 0.2 mM, respectively. Isolated bovine liver branchedchain a-ketoacid dehydrogenase was also inhibited by clofibric acid and phenylpyruvate with ISo values of 0.24 and 0.4 mM, respectively, relative to 0.1 mM a-ketoisovalerate (22). However, the type of inhibition seen with both compounds was of a mixed type (22). While differences in regard to type of inhibition between this study and the previous study (22) may be due to either animal or experimental differences, the relative sensitivity of the complex to inhibition of clofibric acid and phenylpyruvate is the same, with clofibric acid > phenylpyruvate. rnM ((Y -
1 I
FIG. 3. Activation (A, values) of branched-chain cY-ketoacid dehydrogenase in perfused rat hearts by clofibric acid (A), phenylpyruvate (B), and dichloroacetate (C). Perfusion of hearts, extraction of the complex, and determinations of the percentage active form of the complex was as given under Experimental Procedures. Values given are means f SE with 3 hearts for each point except 12 for no additions, 4 for 0.25 mM of each compound, and 6 for 0.5 mM phenylpyruvate. All points were significantly different from no additions at P < 0.05 based on a two-tailed Student’s t test, except with 25 pM clofibric acid (A).
DISCUSSION
vate the complex in the perfused rat heart strongly suggest that their action in the perfused rat heart was inhibition of the kinase and not activation of the phosphatase. Until the specific phosphatase for branched-chain a-ketoacid dehydrogenase is isolated, however, the possibility that these compounds stimulate the phosphatase cannot be definitely ruled out. Using the same conditions as used to generate the A50 values but with a-keto[l14C]isovalerate, the flux through the complex in the presence of 0.1 mM clofibric acid and 0.32 mM phenylpyruvate (Am values) was stimulated 13- and 19-fold, respectively (Table II). Dichloroacetate (1 mM) has been shown previously to increase flux through the complex in rat hearts perfused under similar conditions (15, 16).
Inhibition of branched-chain a-hmbacid dehydrogenase. Clofibric acid was a linear, noncompetitive inhibitor relative to a-ketoisovalerate with an apparent Ki of 0.043
Clofibric acid, phenylpyruvate, and dichloroacetate effects on isolated branchedchain cY-ketoacid dehydrogenase kinase complex suggest the possibility of opposing
TABLE
II
EFFECTS OF CLOFIBRICACID AND PHEN~LPYRUVATE ON (u-KET@~-%]ISOVALERATE OXIDATION BY PERFUSED RAT HEARTS'
Addition
(mM)
None Clofibric acid (0.1) Phenylpyruvate (0.32)
Branched-chain a-ketoacid dehydrogenase flux (nmol min-’ g wet wt-‘) 5.1 * 1.0 (3) 66.5 + 4.7 (6)* 98.4 + 11.4 (5)*
“Perfusions and flux measurements as given under Experimental Procedures. Values are given as means + SE (number of hearts). * Means are significantly different from no additions at P < 0.05 based on a two-tailed Student’s t test.
INHIBITORS
OF
BRANCHED-CHAIN
a-KETOACID
FIG. 4. Inhibition of branched-chain a-ketoacid dehydrogenase by clofibrie acid (A) and phenylpyruvate (B). The complex (1.48 pg) was assayed under the conditions described under Experimental Procedures with various concentrations of either cloflbric acid (A) or phenylpyruvate (B) and o-ketoisovalerate as indicated in the figure. Velocity is given as amol min-’ mg protein-‘. Data are shown as a Lineweaver-Burk plot with the insert showing a replot of either the slope and/or 1/ intercept versus inhibitor concentration.
influences on flux through the complex; inhibition (either mixed or competitive) in regard to substrate utilization, and stimulation through activation (i.e., inhibition of the kinase) of the complex. The overall effect of these compounds on flux through the complex will depend on the intramitochondrial concentration of numerous physiological substrates, modulators (e.g., ADP) and inhibitors of the complex, and on the phosphorylation state of the com-
DEHYDROGENASE
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63
plex. The data obtained with the isolated complex in regard to inhibition of the complex and its associated kinase, which demonstrates and characterizes the direct effect of these compounds, are relative to the substrate (either a-ketoacids or ATP) concentration utilized and may not be absolutely predictive of in vivo effects since the intramitochondrial free concentration of the substrate(s) and inhibitors are not known. The relative potency of these inhibitors, however, was the same when comparing AW values obtained with perfused rat heart with Ido values obtained with the isolated complex (Figs. 2 and 3; see (17)). Studies using isolated hepatocytes with increasing concentrations of dichloroacetate or a-chloroisocaproate showed that, at lower concentration of these compounds, there was an increased flux through the complex due to activation (i.e., inhibition of kinase), while at higher concentrations there was a decreased flux due to direct inhibition of the complex relative to substrate utilization (data not shown). Thus, the large stimulation of flux through the complex seen with clofibric acid and phenylpyruvate in perfused rat heart (Table I) may have been greatly reduced if the phosphorylation state of the complex in the control hearts was greatly reduced (i.e., percentage active form greatly increased). However, under normal physiological conditions the percentage active form of the complex is not very high in nonhepatic tissues (23, 24), and the therapeutic or physiological concentrations of these compounds are similar to the AW concentrations. Thus, the physiological action of these compounds on branched-chain amino acid metabolism is suggested to be mainly on inhibiting the branched-chain a-ketoacid dehydrogenase kinase with subsequent activation of and increased flux through the complex. Clofibric acid inhibited isolated branchedchain cY-ketoacid dehydrogenase kinase and activated the complex in perfused rat heart in the range of 0.05 to 0.5 mM with an AM of 0.1 mM. Clofibric acid at the AW concentration also promoted increased flux through the complex in perfused rat heart. Blood concentration for therapeutic clofi-
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brie acid treatment in humans is between 0.25 and 0.45 mM (25), which is in the effective range for activation of the complex via inhibition of its kinase. Chronic clofibrate treatment can also promote numerous other physiological effects that could also influence branched-chain amino acid metabolism (26-32). However, activation of branched-chain cu-ketoacid dehydrogenase by clofibric acid may, at least partly, be the mechanism whereby clofibrate treatment leads to reduced concentration of blood branched chain amino acid concentrations in humans (10) and rats (ll), increased in vivo Leu oxidation in rats (11) and Tetrahywwmu pgricfomzis (33), and increased branched-chain cy-ketoacid dehydrogenase activity in rat liver (34) and skeletal muscle (11). While all of the above studies showed stimulation of branched-chain amino acid oxidation by clofibric acid, cultured muscle cells show reduced Leu oxidation in the presence of clofibric acid (35-37), with an ISo from 0.25 to 0.85 mM. Another study using hemidiaphragms showed that clofibrie acid (2 mM) inhibited Leu (3 mM) but not Val transamination and decarboxylation (38). Val decarboxylation was only 31% of that seen with Leu (without clofibric acid). The difference between Val and Leu flux through branched-chain a-ketoacid dehydrogenase is opposite to that seen with the isolated complex where a-ketoisovalerate has a greater V,,, than cu-ketoisocaproate (3). These data may be explained in the light of the present work and (17), by the activation state of the complex as influenced by culturing of muscle cells, and by the presence of cu-ketoisocaproate from Leu. In both cases, if the complex is fully activated (i.e., dephosphorylated), then clofibric acid may directly inhibit the flux through the complex by competing with the substrate. Phenylpyruvate inhibited isolated branched-chain cu-ketoacid dehydrogenase and its associated kinase. Phenylpyruvate (Am = 0.32 mM) also activated the complex in perfused rat heart. Phenylpyruvate at the AS0 concentration also increased a-ketoisovalerate oxidation in perfused rat heart. Phenylpyruvate, phenyllactate,
HARRIS
phenylacetate, and o-hydroxyphenylpyruvate are all known to accumulate in phenylketonuria (19) and all can inhibit the isolated branched-chain a-ketoacid dehydrogenase kinase, although phenylpyruvate is the most effective. The Ah0 concentration of phenylpyruvate is within the range of phenylpyruvate seen in untreated phenylketonuric patients, between 0.1 and 0.4 mM (12, 19, 39). When the other abnormal metabolites (e.g., phenyllactate and o-hydroxyphenylacetate) associated with phenylketonuria are added together the total concentration of these compounds would even be higher (14). Inhibition of the kinase may be the mechanism whereby branched-chain amino acid levels are reduced with pathological levels of phenylpyruvate (13). While numerous enzymes are inhibited by phenylpyruvate (40), inhibition is generally observed only at very high phenylpyruvate concentrations. For example, pyruvate dehydrogenase has been shown either not to be inhibited by phenylpyruvate (41) or inhibited uncompetitively at extremely high levels (42). Pyruvate dehydrogenase kinase has also been reported either not to be inhibited by phenylpyruvate at 0.5 mM (43) or inhibited by phenylpyruvate with an I50 value of approximately 2 mM with 10 j&M ATP (42). Thus, branched-chain a-ketoacid dehydrogenase kinase appears to be very sensitive to phenylpyruvate inhibition relative to other enzymes that have been studied, particularly pyruvate dehydrogenase kinase. The role that altered branched-chain amino acid metabolism might have on the pathogenic state seen with phenylketonuria is not clear. However, several studies using rats injected with phenylalanine plus an inhibitor of phenylalanine hydroxylase (LYmethylphenylalanine) have shown that this treatment, which is a model system for phenylketonuria, leads to a correlation between reduced levels of brain branchedchain amino acids with reduced polypeptide elongation and initiation in the brain (44, 45). Dichloroacetate inhibited branched-chain cY-ketoacid dehydrogenase kinase with an Ido value approximately 20 times higher
INHIBITORS
OF
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a-KETOACID
DEHYDROGENASE
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65
than the Id,, value for pyruvate dehydrodehydrogenase and pyruvate dehydrogegenase kinase (3). Dichloroacetate also innase by permitting selective activation of creases Leu oxidation and branched-chain each complex separately and then together. cu-ketoacid dehydrogenase activity in perfused rat hearts (15, 16); and reduces ““Pi ACKNOWLEDGMENTS incorporation into the a-subunit of the CYketoacid decarboxylase component of the We wish to thank Richard Landry for technical complex from rat liver mitochondria (46) assistance, Dr. Arthur Schulz for helpful discussion, and Peggy Smith for expert typing of the manuscript. and rat heart mitochondria (21). At the lowest concentration of dichloroacetate used with the perfused rat heart in this REFERENCES study, 0.25 mM, there was a significant increase in the percentage active form of the 1. KREBS, H. A., ANDLUND, P. (1977) Advan Enzyme complex. The AM value for dichloroacetate Reg. 15,375-394. 2. PARKER, P. J., AND RANDLE, P. J. (1978) B&hem with perfused rat heart was 0.63 mr+h for J 171,751-757. branched-chain a-ketoacid dehydrogenase, 3. PAXTON, R., AND HARRIS, R. A. (1982) J. Biol Ch.em while for pyruvate dehydrogenase the AM 257,14433-14439. value obtained under similar conditions J. 204,353-356. 4. ODESSEY, R. (1982) Biochem. was 0.1 mM (8, 9). The A50 values for py5. FATANIA, H. R., LAU, K. S., AND RANDLE, P. J. ruvate dehydrogenase and branched-chain (1981) FEBS Leti 132, 285-288. ar-ketoacid dehydrogenase are within the 6. HARRIS, R. A., PAXTON, R., AND DEPAOLI-ROACH, therapeutic dichloroacetate blood concenA. A. (1982) J. Biol. Cfiem. 257, 13915-13918. trations of 0.1 to 2 mM with 1 mM (in hu7. HARRIS, R. A., PAXTON, R., AND PARKER, R. A. (1982) Biochem. Biophys. Res. Cwrnmun 107, mans), the maximum effective concentra1497-1503. tion for reducing blood lactate and alaS., AND RANDLE, P. J. (1973) 8. WHITEHOUSE, nine (47). Bio&em. J. 134.651-653. Besides offering a possible mechanism 9. WHITEHOUSE, S., COOPER, R. H., AND RANDLE, of action that these compounds have on P. J. (1974) Biochem J. 141, 761-774. branched-chain amino acid metabolism, 10. WOLFE, B. M., KANE, J. P., HAVEL, R. J., AND the identification and quantitation of their BREWSTER, H. P. (1973) J. CZin Invest. 52,2146inhibitory abilities on branched-chain (Y2159. ketoacid dehydrogenase kinase will also be 11. PAUL, H. S., AND ADIBI, S. A. (1980) J. Clin Invest useful as a means to selectively study the 65,1285-1293. 12. JERVIS, G. A., AND DREJZA, E. J. (1966) Clin Chim. influence of phosphorylation of branchedActa 13. 435-441. chain cu-ketoacid dehydrogenase indepen13. EFRON, M. L., KANG, E. S., VISAKORPI, J., AND dently of pyruvate dehydrogenase. Thus, FELLERS, F. X. (1969) J. Pediat. 74, 399-405. dichloroacetate at very low levels, i.e., 0.0514. CARVER, M. J. (1965) J. Neurochem, 12.45-50. 0.1 mM, should lead to greatly reduced 15. SANS, R. M., JOLLY, W. W., AND HARRIS, R. A. phosphorylation of pyruvate dehydroge(1980) Arch Biochem Biuphys. 200, 336-345. nase with little direct effect on the phos16. SANS, R. M., JOLLY, W. W., AND HARRIS, R. A. phorylation state of branched-chain a-ke(1980) J. Mel CeU Curdiol. 12, 1-16. toacid dehydrogenase. Phenylpyruvate 17. PAXTON, R., AND HARRIS, R. A. (1984) Arch. (0.32 mM) would reduce the phosphorylaBiochem Biophys. 231,46-55. 18. ZAR, J. H. (1974) Biostatistical Analysis, Prenticetion state of branched-chain cz-ketoacid Hall, Englewood Cliffs, New Jersey. dehydrogenase without directly affecting 19. BREMER, H. J., DURAN, M., KAMERLING, J. P., the phosphorylation state of pyruvate dePRZYREMBEL, H., AND WADMAN, S. K. (1981) hydrogenase. Similarly, branched-chain aDisturbances of Amino Acid Metabolism: Clinketoacid dehydrogenase kinase is -90-fold ical Chemistry and Diagnosis, Urban and more sensitive to cY-chloroisocaproate inSchwarzenberg, Baltimore, Maryland. hibition in vitro than pyruvate dehydro20. WAYMACK, P. P., DEBUYSERE, M. S., AND OLSON, genase kinase (6). These agents may lead M. S. (1980) J. Bill Chem 255,9773-9781. to a greater understanding of the inter21. BUXTON, D. B., AND OLSON, M. S. (1982) J. Biol. Chem. 257, 15026-15029. action between branched-chain cY-ketoacid
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22. DANNER, D. J., SEWELL, E. T., AND ELSAS, L. J. (1982) J. Biol Chem 257, 659-662. 23. GILLIM, S. E., PAXTON, R., Coo& G. A., AND HARRIS, R. A. (1983) Biochem. Biwphys. Res. Cmmun. 111,74-81. 24. ODESSEY, R. (1980) Biochem J. 192,155-X3. 25. D’COSTA, M. A., SMIGURA, F. C., KULHAY, K., AND ANGEL, A. (1977) J. Lab. Clin Med 90,823-835. 26. RUEGAMER, W. R., RYAN, N. T., RICHERT, D. A., AND WESTERFELD, W. W. (1969) Biochem Pharmmol 18, 613-624. 27. PAUL, H. S., AND ADIBI, S. A. (19’79) J. C&n. Invest. 64.405-412. 28. KURUP, C. K. R., AITHAL, H. N., AND RAMASARMA, T. (1970) B&hem J. 116,773-779. 29. LAZAROW, P. B. (1977) Science 197,580-581. 30. VOLTTI, H., SAVOLAINEN, M. J., JAUHONEN, V. P., AND HASSINEN, I. E. (1979) Biochem J. 182,95102. 31. REDDY, J. K., AND KUMAR, N. S. (1979) J. Biochem 85,847-856. 32. KERNER, J., AND BIEBER, L. L. (1982) Biochem Med 28,204-209. 33. BLUMM, J. J. (1980) B&him Biophys. Acta 628, 46-56. 34. PAUL, H. S., AND ADIBI, S. A. (1982) J. Biol Chem+ 257,4875-4881. 35. PARDRIDGE, W. M., CASANELLO-ERTL, D., AND DUDUCGIAN-VARTAVARIAN, L. (1980) J. Clin Invest. 66,88-93.
HARRIS 36. PARDRIDGE, W. M. (1981) in Metabolism and Clinical Implications of Branched-Chain Amino and Keto Acids (Walser, M., and Williamson, J. R., eds.), pp. 263-269, Elsevier/North-Holland, New York/Amsterdam. 37. PARDRIDGE, W. M., DUDUCGIAN-VARTAVARIAN, L., CASANELLO-ERTL, D., JONES, M. R., AND KOPPLE, J. D. (1981) Amer. J. Physid 240, E203-E208. 38. TROKE, J. M., DUFF, D. A., AND SNELL, K. (1983) Biochem Sot. Tram 11,289~290. 39. PATEL, M. S. (1972) Biochem J. 128,677-684. 40. LAND, J. M., AND CLARK, J. B. (1973) Biochem J. 134, 545-555. 41. LAND, J. M., AND CLARK, J. B. (1973) B&hem J 134,539-544. 42. HOFFMANN, B. T., AND HUCHO, F. (1974) FEBS Z&t. 43, 116-119. 43. DENTON, R. M., RANDLE, P. J., BRIDGES, B. J., COOPER, R. H., KERBEY, A. L., PASK, H. T., SEVERSON, D. L., STANSBIE, D., AND WHITEHOUSE, S. (1973) MoL Cell Biochem 9,27-53. 44. HUGHES, J. V., AND JOHNSON, T. C. (1978) Biochim Biophys. Acta 517, 473-485. 45. BINEK-SINGER, P., AND JOHNSON, T. C. (1982) Biochxem. J. 206, 407-414. 46. HUGHES, W. A., AND HALESTRAP, A. P. (1981) Biochem. J. 196,459-469. 47. WELLS, P. G., MOORE, G. W., RABIN, D., W~XINSON, G. R., OATES, J. A., AND~TACPOOLE, P. W. (1980) &&to&a 19,109-113.
OF BIOCHEMISTRY
Vol. 231, No. 1, May
AND BIOPHYSICS 15, pp. 58-66, 1984
Clofibric Acid, Phenylpyruvate, and Dichloroacetate Branched-Chain cw-Ketoacid Dehydrogenase in Vitro and in Perfused Rat Heart’ RALPH of Bicdemistry,
Department
Received
PAXTON Indiana
September
AND
University
ROBERT School
‘7, 1983, and in revised
Inhibition of Kinase
A. HARRIS’
of Medicine, form
Indianapolis,
January
Indiana
46223
23, 1984
Branched-chain a-ketoacid dehydrogenase kinase, purified from rabbit liver, was inhibited by clofibric acid, phenylpyruvate, and dichloroacetate in a mixed manner relative to ATP. 14,,values relative to 75 ELM ATP were 0.33,1.7, and 3.0 mM, respectively. Inhibition of the kinase by acetate, pyruvate, and lactate was minimal; whereas a phydroxyphenyl substitution of these compounds increased their potency as kinase inhibitors, a phenyl substitution gave the most potent inhibitors. Clofibric acid, phenylpyruvate, and dichloroacetate activated branched-chain a-ketoacid dehydrogenase in perfused rat hearts. Perfusate concentrations that gave 50% activation (A& were 0.1, 0.32, and 0.63 mM, respectively. A50 concentrations of clofibric acid and phenylpyruvate also increased flux (decarboxylation of a-keto[l-‘4C]isovalerate) through branched-chain cu-ketoacid dehydrogenase in perfused rat heart. These findings suggest that, although clofibric acid and phenylpyruvate can inhibit substrate utilization by the branchedchain cu-ketoacid dehydrogenase complex, the major effect of these compounds on branched-chain amino acid metabolism is due to inhibition of branched-chain a-ketoacid dehydrogenase kinase with subsequent activation of and increased flux through the complex.
Branched-chain a-ketoacid dehydrogenase complex (EC 1.2.4.4) is a mitochondrial multienzyme complex that catalyzes the oxidative decarboxylation of cy-ketoisocaproate, a-ketoisovalerate, and a-ketofi-methylvalerate (transamination products of Leu, Val, and Ile, respectively). The complex is rate limiting for the metabolism of branched-chain amino acids, except in liver where transamination is rate limiting (1). Activity of the complex is regulated
competitively by product inhibition (2) and covalently through phosphorylation, which decreases V,,, (3-5). The phosphorylation state of the complex will be determined by the combined actions of a kinase, which has been partially characterized (3), and by a phosphatase that has not yet been isolated or characterized. Thus, branchedchain amino acid metabolism will be determined by the flux through the complex which is regulated at two levels, with the maximum flux (i.e., V,,,) dependent upon the phosphorylation state (i.e., relative kinase to phosphatase activity) and modulation of this maximum flux by substrate and product influences. Alteration of phosphorylation state through kinase inhibition has been shown for two mitochondrial multienzyme complexes (i.e., branched-chain Lu-ketoacid de-
i This work was supported in part by NIH Research Grants AM19259 and 5 SO7 RR5371, and the Grace M. Showalter Residuary Trust. A preliminary report of this work was presented at the 1983 meeting of the American Society of Biological Chemists in San Francisco, Calif. ‘To whom correspondence and reprint requests should be addressed. 0003-9861/84 Copyright All rights
$3.00
0 1984 by Academic Press, Inc. of reproduction in any form reserved.
58
INHIBITORS
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59
hydrogenase and pyruvate dehydrogenase, concentration of these compounds needed EC 1.2.4.1) to decrease the phosphorylation for these effects in perfused rat heart is state (increased percentage active form) similar to therapeutic or physiological concentrations, the effects of these comand to increase the flux through these pounds on branched-chain amino acids complexes. cr-Chloroisocaproate inhibits isolated branched-chain ar-ketoacid dehymay be mediated through activation of the complex. drogenase kinase, and decreases the phosphorylation state of and increases the flux through the complex in perfused rat heart EXPERIMENTAL PROCEDURES (6, 7). Similarly, dichloroacetate inhibits isolated pyruvate dehydrogenase kinase, Materials. Reagents were obtained as previously and decreases the phosphorylation state of given (3, 6, 17), with the following exceptions: aand increases the flux through this complex Kewl-“Cjisovalerate was from Amersham Corp.; and in perfused rat heart and other tissues (8, dichloroacetic acid and Scinti Verse II were from 9). Thus, alterations in phosphorylation Fisher Scientific Company. cr-Chloroisocaproate was a gift from Dr. Robert J. Strohscheim and Dr. Ronald state of these complexes, which influences Simpson of Sandoz, Inc. V max9 appears to determine maximum flux Branched-chain &Aoacid dehydrogenase isolatiolz under a given set of conditions. and assay. Rabbit liver branched-chain cy-ketoacid Several compounds of medical or biodehydrogenase with intrinsic kinase activity was isochemical interest affect branched-chain lated and assayed as given in (17). The spectrophoamino acid metabolism and may therefore tometric assay of the complex was done at 30°C as influence the phosphorylation state of previously described (3), with other conditions as given branched-chain a-ketoacid dehydrogenase. in the text. Clofibric acid (2-(4-chlorophenoxy)-2Branched-chain a-ketoacid dehydrogenase kinase methylpropionic acid), the active agent of assay. Branched-chain a-ketoacid dehydrogenase ki32P as previously clofibrate (ethyl ester of clofibric acid), is nase was assayed by protein-bound described (17). an antihyperlipidemic and antihyperchoIsolation of broa&specifcity protein phosphutase. lesterolemic agent that also reduces blood Broad-specificity protein phosphatase isolation and branched-chain amino acids concentration assay was as previously described (17). in humans (10) and rats (11). PhenylkeHeart pmfusia studies. Hearts from 300- to 500tonuria, an inherited metabolic disorder g male Wistar rats were perfused by the flow-through involving phenylalanine metabolism, leads Langendorff procedure as previously described (17). to appearance of phenylpyruvate in the Hearts were perfused at 37°C for the indicated time blood (12) and reduced concentration of with Krebs-Henseleit buffer containing 10 mM glublood branched-chain amino acids (13). cose, 20 mu/ml bovine insulin, 0.1 mM cY-ketoisovalSimilarly, a large dose of phenylalanine erate (which had a specific activity of 30 to 50 dpm/ nmol of cu-keto[l-%]isovalerate for branched-chain administered to rats leads to reduced concu-ketoacid dehydrogenase flux measurement), and centrations of blood and liver branchedadditions as given in the test. chain amino acids (14). Dichloroacetate inDetermination of percentage active from branchedhibits isolated rabbit liver branched-chain chain cu-ketoacid dehydrogenase in perfused rat heart. cy-ketoacid dehydrogenase kinase (3) and of the percentage active form of increases flux through branched-chain (Y- The determination the complex was as previously given (17). ketoacid dehydrogenase in perfused rat Branched-chain ol-ketoacid dehydrogenase j&x hearts (15,16). However, the sensitivity of measurements in perfused rat heart. Hearts were perthe complex to activation by dichloroacefused as above (with a-keto[l-‘“Clisovalerate) for 15 tate has not been determined in perfused min and then additions to perfusate were made as rat heart. given. At 40 min from the start of perfusion the perThe results of this study demonstrate fusate from the heart was collected for 1 min in a that clofibric acid and phenylpyruvate can 25-ml Erlenmeyer flask that contained 0.5 mmol KOH inhibit isolated branched-chain a-ketoacid and 50 pmol EDTA. The collected perfusate volume dehydrogenase kinase, activate the comwas determined gravimetrically and the flask was plex, and increase the flux through the fitted with a serum cap containing a plastic center well. The collected perfusate was then acidified with complex in perfused rat heart. Since the
60
PAXTON
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0.3 ml of 0.8 M KHzPOl and 3.5 M H,PO,. Liberated COP was collected in the center well which contained 0.3 ml of 1 M KOH. After 1.5 to 2 h in a shaking water bath at 20°C the center wells were removed and placed in ScintiVerse II, and radioactivity was determined. Kinetic analyses were performed as previously given (3). All statistical tests and associated tables were from Ref. (18).
RESULTS
Inhibition of branched-chain cu-ketoacid dehydrogenase kinase by cloJibric acid, phenylpymvate, and dichloroacetate. Clofibric acid (Fig. lA), phenylpyruvate (Fig. lB), and dichloroacetate (data not shown) showed mixed inhibition of branched-chain a-ketoacid dehydrogenase kinase relative to ATP. The exact mechanism of inhibition was not clear since the reciprocal plots of velocity versus ATP concentrations did not have a common intersection and the replots of the y intercepts versus inhibitor concentration were apparently nonlinear. Since the mechanism of inhibition was not known the quantitation of the relative potency of these compounds to inhibit the kinase could not be determined from these data. However, readily comparable values of inhibitor potency were determined with a single ATP concentration (75 PM). IbOvalues (amount of compound needed to inhibit 40% of total branched-chain a-ketoacid dehydrogenase kinase activity; Fig. 2) were selected as the comparison value since the inhibition was nonlinear and it appeared that the inhibition pattern at 40% was fairly uniform between the different compounds. The inhibitor concentrations needed to achieve 40% inhibition (IU value) of the total branched-chain a-ketoacid dehydrogenase kinase activity were 0.33,1.7, and 3.0 mM for clofibric acid, phenylpyruvate, and dichloroacetate, respectively (Fig. 2). It should be noted that these estimates of the relative potency of these compounds to inhibit the kinase, although not independent of ATP concentration, were found to correspond to the relative abilities of these compounds to activate the complex in perfused rat heart as shown below.
FIG. 1. Inhibition of branched-chain cY-ketoacid dehydrogenase kinase by clofibric acid (A) and phenylpyruvate (B). Branched-chain cY-ketoacid dehydrogenase kinase was assayed under the conditions described under Experimental Procedures, with various concentrations of either clofibric acid (A) or phenylpyruvate (B) and ATP as indicated in the figure. Velocity is nmol 9 bound 20 min? mg protein-‘.
Since elevated phenylpyruvate levels associated with phenylketonuria are also associated with elevated levels of other abnormal metabolites of phenylpyruvate (e.g. phenylacetate and phenyllactate (19)) several other compounds associated with phenylketonuria were also tested as inhibitors of branched-chain cu-ketoacid dehydrogenase kinase (Table I). While acetate, pyruvate, and lactate had minimal inhibitory effects, addition of a phenyl
INHIBITORS
OF
BRANCHED-CHAIN
a-KETOACID
FIG. 2. Inhibition (I, values) of branched-chain cY-ketoacid dehydrogenase kinase by dichloroacetate (A), phenylpyruvate (B), and clofibric acid (C). Branched-chain a-ketoacid dehydrogenase kinase was assayed as in Fig. 1, except assay time was 30 min and ATP concentration was 75 PM with other additions as given in the figure. Under these conditions 100% branched-chain cY-ketoacid dehydrogenase kinase activity (nmol 32p bound 30 min-’ mg protein-‘) was 5.13 f 0.13 (it + SE) for 33 determinations.
DEHYDROGENASE
and 4.3% f 1.0 (12) in the presence of (Yketoisovalerate. The total activity (nmol min-’ g wet wt-’ at 30°C with 1 mM (Yketoisovalerate) for 84 hearts done in a similar manner (combined number of hearts in this study and another (17)) was 248 f 40 (ii f SD). Since under these conditions the complex must be almost completely phosphorylatecl, any compound that selectively inhibits branched chain cz-ketoacid dehyclrogenase kinase should result in an elevated percentage active form of and flux through the complex. The percentage active form of branchedchain a-ketoacid dehydrogenase in perfused rat heart was increased with increasing concentrations of clofibric acid, phenylpyruvate, and dichloroacetate (Fig. 3). The concentration of clofibric acid, phenylpyruvate, and dichloroacetate needed to achieve 50% activation (Am value) was 0.1,0.32, and 0.63 mM, respectively. It cannot be excluded that these compounds activated the complex by stimulation of the branched-chain a-ketoacid dehydrogenase phosphatase. However, the fact that these compounds inhibited the isolated branchedchain a-ketoacid dehydrogenase kinase and that the relative inhibitory abilities of these compounds to inhibit the isolated kinase are similar to their abilities to actiTABLE
group greatly increased potency of these carboxylic acids to inhibit branched-chain a-ketoacicl dehydrogenase kinase. However, a p-hydroxy substitution on the phenyl group reduced the potency of these compounds. Phenylalanine (2 mM) did not inhibit the kinase (data not shown). Activation of branched-chain a-h&x&d dehydrogenase in perfused rat heart by clujibric acid, phen&yruvate, and dichbm acetate. Rat hearts perfused with glucose have a very low activity of and flux through branched-chain a-ketoacid dehydrogenase (this study and (15, 16, 20, 21)). The presence of 0.1 mM cY-ketoisovalerate did not change the percentage active form of branched-chain a-ketoacid dehydrogenase in perfused rat hearts with ii + SE [number of hearts] of 3.1% + 0.6 (3) in the absence
61
KINASE
I
EFFECTS OF VARIOUS COMPOUNDS ON INHIBITION BRANCHED-CHAIN PKETOACID DEHYDROGENASE’
Compound
Percentage inhibition
(2 mM)
Acetate Phenylacetate o-Hydroxyphenylacetate pHydroxyphenylacetate
15 32 6 45
Pyruvate Phenylpyruvate p-hydroxyphenylpyruvate
8 50 30
Lactate Phenyllactate pHydroxyphenyllactate
0 24 12
“Determined as given centration was 0.2 mM.
OF
in Fig.
2 except
ATP
con-
PAXTON
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3.3), while phenylpyruvate was competitive (Fig. 4B) with an apparent Ki of 0.05 mM. No qualitative difference in the type of inhibition has been seen when different branched-chain a-ketoacids were used to analyze cY-chloroisocaproate inhibition (6), and therefore the use of any LYketoacid substrate appears to adequately describe the type of inhibition. Ir,,, values (50% inhibition of total activity) with 56 PM a-ketoisovalerate for clofibric acid and phenylpyruvate were 0.08 and 0.2 mM, respectively. Isolated bovine liver branchedchain a-ketoacid dehydrogenase was also inhibited by clofibric acid and phenylpyruvate with ISo values of 0.24 and 0.4 mM, respectively, relative to 0.1 mM a-ketoisovalerate (22). However, the type of inhibition seen with both compounds was of a mixed type (22). While differences in regard to type of inhibition between this study and the previous study (22) may be due to either animal or experimental differences, the relative sensitivity of the complex to inhibition of clofibric acid and phenylpyruvate is the same, with clofibric acid > phenylpyruvate. rnM ((Y -
1 I
FIG. 3. Activation (A, values) of branched-chain cY-ketoacid dehydrogenase in perfused rat hearts by clofibric acid (A), phenylpyruvate (B), and dichloroacetate (C). Perfusion of hearts, extraction of the complex, and determinations of the percentage active form of the complex was as given under Experimental Procedures. Values given are means f SE with 3 hearts for each point except 12 for no additions, 4 for 0.25 mM of each compound, and 6 for 0.5 mM phenylpyruvate. All points were significantly different from no additions at P < 0.05 based on a two-tailed Student’s t test, except with 25 pM clofibric acid (A).
DISCUSSION
vate the complex in the perfused rat heart strongly suggest that their action in the perfused rat heart was inhibition of the kinase and not activation of the phosphatase. Until the specific phosphatase for branched-chain a-ketoacid dehydrogenase is isolated, however, the possibility that these compounds stimulate the phosphatase cannot be definitely ruled out. Using the same conditions as used to generate the A50 values but with a-keto[l14C]isovalerate, the flux through the complex in the presence of 0.1 mM clofibric acid and 0.32 mM phenylpyruvate (Am values) was stimulated 13- and 19-fold, respectively (Table II). Dichloroacetate (1 mM) has been shown previously to increase flux through the complex in rat hearts perfused under similar conditions (15, 16).
Inhibition of branched-chain a-hmbacid dehydrogenase. Clofibric acid was a linear, noncompetitive inhibitor relative to a-ketoisovalerate with an apparent Ki of 0.043
Clofibric acid, phenylpyruvate, and dichloroacetate effects on isolated branchedchain cY-ketoacid dehydrogenase kinase complex suggest the possibility of opposing
TABLE
II
EFFECTS OF CLOFIBRICACID AND PHEN~LPYRUVATE ON (u-KET@~-%]ISOVALERATE OXIDATION BY PERFUSED RAT HEARTS'
Addition
(mM)
None Clofibric acid (0.1) Phenylpyruvate (0.32)
Branched-chain a-ketoacid dehydrogenase flux (nmol min-’ g wet wt-‘) 5.1 * 1.0 (3) 66.5 + 4.7 (6)* 98.4 + 11.4 (5)*
“Perfusions and flux measurements as given under Experimental Procedures. Values are given as means + SE (number of hearts). * Means are significantly different from no additions at P < 0.05 based on a two-tailed Student’s t test.
INHIBITORS
OF
BRANCHED-CHAIN
a-KETOACID
FIG. 4. Inhibition of branched-chain a-ketoacid dehydrogenase by clofibrie acid (A) and phenylpyruvate (B). The complex (1.48 pg) was assayed under the conditions described under Experimental Procedures with various concentrations of either cloflbric acid (A) or phenylpyruvate (B) and o-ketoisovalerate as indicated in the figure. Velocity is given as amol min-’ mg protein-‘. Data are shown as a Lineweaver-Burk plot with the insert showing a replot of either the slope and/or 1/ intercept versus inhibitor concentration.
influences on flux through the complex; inhibition (either mixed or competitive) in regard to substrate utilization, and stimulation through activation (i.e., inhibition of the kinase) of the complex. The overall effect of these compounds on flux through the complex will depend on the intramitochondrial concentration of numerous physiological substrates, modulators (e.g., ADP) and inhibitors of the complex, and on the phosphorylation state of the com-
DEHYDROGENASE
KINASE
63
plex. The data obtained with the isolated complex in regard to inhibition of the complex and its associated kinase, which demonstrates and characterizes the direct effect of these compounds, are relative to the substrate (either a-ketoacids or ATP) concentration utilized and may not be absolutely predictive of in vivo effects since the intramitochondrial free concentration of the substrate(s) and inhibitors are not known. The relative potency of these inhibitors, however, was the same when comparing AW values obtained with perfused rat heart with Ido values obtained with the isolated complex (Figs. 2 and 3; see (17)). Studies using isolated hepatocytes with increasing concentrations of dichloroacetate or a-chloroisocaproate showed that, at lower concentration of these compounds, there was an increased flux through the complex due to activation (i.e., inhibition of kinase), while at higher concentrations there was a decreased flux due to direct inhibition of the complex relative to substrate utilization (data not shown). Thus, the large stimulation of flux through the complex seen with clofibric acid and phenylpyruvate in perfused rat heart (Table I) may have been greatly reduced if the phosphorylation state of the complex in the control hearts was greatly reduced (i.e., percentage active form greatly increased). However, under normal physiological conditions the percentage active form of the complex is not very high in nonhepatic tissues (23, 24), and the therapeutic or physiological concentrations of these compounds are similar to the AW concentrations. Thus, the physiological action of these compounds on branched-chain amino acid metabolism is suggested to be mainly on inhibiting the branched-chain a-ketoacid dehydrogenase kinase with subsequent activation of and increased flux through the complex. Clofibric acid inhibited isolated branchedchain cY-ketoacid dehydrogenase kinase and activated the complex in perfused rat heart in the range of 0.05 to 0.5 mM with an AM of 0.1 mM. Clofibric acid at the AW concentration also promoted increased flux through the complex in perfused rat heart. Blood concentration for therapeutic clofi-
64
PAXTON
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brie acid treatment in humans is between 0.25 and 0.45 mM (25), which is in the effective range for activation of the complex via inhibition of its kinase. Chronic clofibrate treatment can also promote numerous other physiological effects that could also influence branched-chain amino acid metabolism (26-32). However, activation of branched-chain cu-ketoacid dehydrogenase by clofibric acid may, at least partly, be the mechanism whereby clofibrate treatment leads to reduced concentration of blood branched chain amino acid concentrations in humans (10) and rats (ll), increased in vivo Leu oxidation in rats (11) and Tetrahywwmu pgricfomzis (33), and increased branched-chain cy-ketoacid dehydrogenase activity in rat liver (34) and skeletal muscle (11). While all of the above studies showed stimulation of branched-chain amino acid oxidation by clofibric acid, cultured muscle cells show reduced Leu oxidation in the presence of clofibric acid (35-37), with an ISo from 0.25 to 0.85 mM. Another study using hemidiaphragms showed that clofibrie acid (2 mM) inhibited Leu (3 mM) but not Val transamination and decarboxylation (38). Val decarboxylation was only 31% of that seen with Leu (without clofibric acid). The difference between Val and Leu flux through branched-chain a-ketoacid dehydrogenase is opposite to that seen with the isolated complex where a-ketoisovalerate has a greater V,,, than cu-ketoisocaproate (3). These data may be explained in the light of the present work and (17), by the activation state of the complex as influenced by culturing of muscle cells, and by the presence of cu-ketoisocaproate from Leu. In both cases, if the complex is fully activated (i.e., dephosphorylated), then clofibric acid may directly inhibit the flux through the complex by competing with the substrate. Phenylpyruvate inhibited isolated branched-chain cu-ketoacid dehydrogenase and its associated kinase. Phenylpyruvate (Am = 0.32 mM) also activated the complex in perfused rat heart. Phenylpyruvate at the AS0 concentration also increased a-ketoisovalerate oxidation in perfused rat heart. Phenylpyruvate, phenyllactate,
HARRIS
phenylacetate, and o-hydroxyphenylpyruvate are all known to accumulate in phenylketonuria (19) and all can inhibit the isolated branched-chain a-ketoacid dehydrogenase kinase, although phenylpyruvate is the most effective. The Ah0 concentration of phenylpyruvate is within the range of phenylpyruvate seen in untreated phenylketonuric patients, between 0.1 and 0.4 mM (12, 19, 39). When the other abnormal metabolites (e.g., phenyllactate and o-hydroxyphenylacetate) associated with phenylketonuria are added together the total concentration of these compounds would even be higher (14). Inhibition of the kinase may be the mechanism whereby branched-chain amino acid levels are reduced with pathological levels of phenylpyruvate (13). While numerous enzymes are inhibited by phenylpyruvate (40), inhibition is generally observed only at very high phenylpyruvate concentrations. For example, pyruvate dehydrogenase has been shown either not to be inhibited by phenylpyruvate (41) or inhibited uncompetitively at extremely high levels (42). Pyruvate dehydrogenase kinase has also been reported either not to be inhibited by phenylpyruvate at 0.5 mM (43) or inhibited by phenylpyruvate with an I50 value of approximately 2 mM with 10 j&M ATP (42). Thus, branched-chain a-ketoacid dehydrogenase kinase appears to be very sensitive to phenylpyruvate inhibition relative to other enzymes that have been studied, particularly pyruvate dehydrogenase kinase. The role that altered branched-chain amino acid metabolism might have on the pathogenic state seen with phenylketonuria is not clear. However, several studies using rats injected with phenylalanine plus an inhibitor of phenylalanine hydroxylase (LYmethylphenylalanine) have shown that this treatment, which is a model system for phenylketonuria, leads to a correlation between reduced levels of brain branchedchain amino acids with reduced polypeptide elongation and initiation in the brain (44, 45). Dichloroacetate inhibited branched-chain cY-ketoacid dehydrogenase kinase with an Ido value approximately 20 times higher
INHIBITORS
OF
BRANCHED-CHAIN
a-KETOACID
DEHYDROGENASE
KINASE
65
than the Id,, value for pyruvate dehydrodehydrogenase and pyruvate dehydrogegenase kinase (3). Dichloroacetate also innase by permitting selective activation of creases Leu oxidation and branched-chain each complex separately and then together. cu-ketoacid dehydrogenase activity in perfused rat hearts (15, 16); and reduces ““Pi ACKNOWLEDGMENTS incorporation into the a-subunit of the CYketoacid decarboxylase component of the We wish to thank Richard Landry for technical complex from rat liver mitochondria (46) assistance, Dr. Arthur Schulz for helpful discussion, and Peggy Smith for expert typing of the manuscript. and rat heart mitochondria (21). At the lowest concentration of dichloroacetate used with the perfused rat heart in this REFERENCES study, 0.25 mM, there was a significant increase in the percentage active form of the 1. KREBS, H. A., ANDLUND, P. (1977) Advan Enzyme complex. The AM value for dichloroacetate Reg. 15,375-394. 2. PARKER, P. J., AND RANDLE, P. J. (1978) B&hem with perfused rat heart was 0.63 mr+h for J 171,751-757. branched-chain a-ketoacid dehydrogenase, 3. PAXTON, R., AND HARRIS, R. A. (1982) J. Biol Ch.em while for pyruvate dehydrogenase the AM 257,14433-14439. value obtained under similar conditions J. 204,353-356. 4. ODESSEY, R. (1982) Biochem. was 0.1 mM (8, 9). The A50 values for py5. FATANIA, H. R., LAU, K. S., AND RANDLE, P. J. ruvate dehydrogenase and branched-chain (1981) FEBS Leti 132, 285-288. ar-ketoacid dehydrogenase are within the 6. HARRIS, R. A., PAXTON, R., AND DEPAOLI-ROACH, therapeutic dichloroacetate blood concenA. A. (1982) J. Biol. Cfiem. 257, 13915-13918. trations of 0.1 to 2 mM with 1 mM (in hu7. HARRIS, R. A., PAXTON, R., AND PARKER, R. A. (1982) Biochem. Biophys. Res. Cwrnmun 107, mans), the maximum effective concentra1497-1503. tion for reducing blood lactate and alaS., AND RANDLE, P. J. (1973) 8. WHITEHOUSE, nine (47). Bio&em. J. 134.651-653. Besides offering a possible mechanism 9. WHITEHOUSE, S., COOPER, R. H., AND RANDLE, of action that these compounds have on P. J. (1974) Biochem J. 141, 761-774. branched-chain amino acid metabolism, 10. WOLFE, B. M., KANE, J. P., HAVEL, R. J., AND the identification and quantitation of their BREWSTER, H. P. (1973) J. CZin Invest. 52,2146inhibitory abilities on branched-chain (Y2159. ketoacid dehydrogenase kinase will also be 11. PAUL, H. S., AND ADIBI, S. A. (1980) J. Clin Invest useful as a means to selectively study the 65,1285-1293. 12. JERVIS, G. A., AND DREJZA, E. J. (1966) Clin Chim. influence of phosphorylation of branchedActa 13. 435-441. chain cu-ketoacid dehydrogenase indepen13. EFRON, M. L., KANG, E. S., VISAKORPI, J., AND dently of pyruvate dehydrogenase. Thus, FELLERS, F. X. (1969) J. Pediat. 74, 399-405. dichloroacetate at very low levels, i.e., 0.0514. CARVER, M. J. (1965) J. Neurochem, 12.45-50. 0.1 mM, should lead to greatly reduced 15. SANS, R. M., JOLLY, W. W., AND HARRIS, R. A. phosphorylation of pyruvate dehydroge(1980) Arch Biochem Biuphys. 200, 336-345. nase with little direct effect on the phos16. SANS, R. M., JOLLY, W. W., AND HARRIS, R. A. phorylation state of branched-chain a-ke(1980) J. Mel CeU Curdiol. 12, 1-16. toacid dehydrogenase. Phenylpyruvate 17. PAXTON, R., AND HARRIS, R. A. (1984) Arch. (0.32 mM) would reduce the phosphorylaBiochem Biophys. 231,46-55. 18. ZAR, J. H. (1974) Biostatistical Analysis, Prenticetion state of branched-chain cz-ketoacid Hall, Englewood Cliffs, New Jersey. dehydrogenase without directly affecting 19. BREMER, H. J., DURAN, M., KAMERLING, J. P., the phosphorylation state of pyruvate dePRZYREMBEL, H., AND WADMAN, S. K. (1981) hydrogenase. Similarly, branched-chain aDisturbances of Amino Acid Metabolism: Clinketoacid dehydrogenase kinase is -90-fold ical Chemistry and Diagnosis, Urban and more sensitive to cY-chloroisocaproate inSchwarzenberg, Baltimore, Maryland. hibition in vitro than pyruvate dehydro20. WAYMACK, P. P., DEBUYSERE, M. S., AND OLSON, genase kinase (6). These agents may lead M. S. (1980) J. Bill Chem 255,9773-9781. to a greater understanding of the inter21. BUXTON, D. B., AND OLSON, M. S. (1982) J. Biol. Chem. 257, 15026-15029. action between branched-chain cY-ketoacid
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