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Regulation of the branched-chain α-ketoacid dehydrogenase and elucidation of a molecular basis for maple syrup urine disease
REGULATION OF THE BRANCHED-CHAIN a-KETOACID DEHYDROGENASE AND ELUCIDATION OF A MOLECULAR BASIS FOR MAPLE SYRUP URINE DISEASE ROBERT A. HARRIS, BEI ZHANG, GARY W. GOODWIN, MARTHA J. KUNTZ, YOSHIHARU SHIMOMURA, PAUL ROUGRAFF, PAUL DEXTER, YU ZHAO, REID GIBSON and DAVID W. CRABB Departments of Biochemistry and Medicine, Indiana University School of Medicine, Indianapolis, Indiana 46202
INTRODUCTION
With the exception of the valine pathway (l), the enzyme-catalyzed reactions for the oxidation of branched-chain amino acids are well established. A high-capacity system for branched-chain amino acid catabolism is needed to prevent severe toxic effects of these amino acids and their metabolites that are characteristic of maple syrup urine disease. Nevertheless, catabolism of branched-chain amino acids must be under tight control, particularly when dietary protein intake is restricted, to assure availability of these essential amino acids for protein synthesis. Various physiological conditions (e.g. starvation, diabetes, trauma) influence BCAA metabolism, attesting to the importance of the regulatory mechanisms for their catabolism. There is much interest in mechanisms that regulate branched-chain amino acid levels because leucine has been shown to inhibit protein degradation (2), stimulate protein synthesis (3), and promote insulin release (4, 5). Although controversial, there is also interest in a possible role of branched-chain amino acids in the pathogenesis and treatment of hepatic encephalopathy (6). Regulation of branched-chain amino acid catabolism is achieved in large part at the branched-chain ol-ketoacid dehydrogenase complex (7-9), the enzyme responsible for oxidative decarboxylation of the a-ketoacids derived by transamination of branched-chain amino acids. This multienzyme complex, composed of an a-ketoacid decarboxylase (El, EC 1.2.4.4), a transacylase (E2, no EC number), and dihydrolipoamide reductase (E3, EC 1.8.1.4), is subject to regulation by covalent modification (l&12). The kinase (no EC number) which phosphorylates the Ela subunit and 245
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inactivates the decarboxylase is also tightly associated with the complex. E1 is composed of two different subunits in an a2132 structure (13); there are 24 of each of the E1 subunits and 24 E2 subunits in the complex (14, 15). The pioneering work of the laboratories of Reed (15) and Danner (16) in purification of the complex opened the door for the study of its regulation. The kinase is inhibited by branched-chain et-ketoacids (17, 18), occhloroisocaproate (19), and dichloroacetate (20). Phosphorylation occurs on two serine residues of the Elot subunit, with site 1 (Ser 293) probably more important than site 2 (Ser 303) in modifying enzyme activity (21, 22). The kinase has neither been purified nor resolved from the complex. In contrast, the kinase (EC2.7.1.99) responsible for phosphorylation of the related pyruvate dehydrogenase complex (EC 1.2.4.1 + EC 2.3.1.12 + EC 1.8.1.4) has been isolated and partially characterized. It has an ot13 structure with subunit molecular weights of 48,000 and 45,000 (23). A protein of 45,000 daltons which may correspond to a kinase subunit has been observed by SDS-polyacrylamide gel electrophoresis with some preparations of the branched-chain otketoacid dehydrogenase complex (12). The branched-chain a-ketoacid dehydrogenase phosphatase (no EC number), also known to be distinct from pyruvate dehydrogenase phosphatase (EC 3.1.3.43), has an apparent M r of 460,000 with a catalytic subunit of 33,000 (24, 25). Regulation of the activity state of the branched-chain ot-ketoacid dehydrogenase in various physiological states has been studied in the laboratories (7-9, 26-31). As summarized below, there is basic agreement with respect to the activity state of the hepatic enzyme in various nutritional states. Restriction of protein intake by feeding diets low in protein results in activation of hepatic branched-chain ot-ketoacid dehydrogenase complex (9, 32), thereby conserving branched-chain amino acids for protein synthesis during periods of protein starvation. Protein intake beyond that needed for maximum growth maintains branched-chain et-ketoacid dehydrogenase complex in the active state, thereby providing a mechanism for disposal of branched-chain amino acids in excess of that required for protein synthesis. The original experiments (33, 34) demonstrating decreased capacity of the liver in rats fed a low-protein diet to oxidize branched-chain keto acids are now readily explained by covalent modification of the branched-chain aketoacid dehydrogenase complex. Starvation of rats previously maintained on chow diet causes no significant change in the activity state of hepatic branched-chain a-ketoacid dehydrogenase complex (9, 32). Starvation of rats previously fed a low-protein (8%) diet, in which the enzyme is initially in a low-activity state, results in activation (9, 32). Inactivation of the complex observed in the protein-deficient state may be due to less
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inhibition of the kinase because of reduced plasma and tissue concentrations of branched-chain ot-ketoacids. No change in activity state is observed upon starvation of chow-fed rats because excess branched-chain ketoacids are supplied to the liver in both cases, i.e. by digestion of dietary protein in the fed state and by endogenous proteolysis in the starved state. Starvation of rats fed low-protein diets promotes endogenous proteolysis, giving rise to increased blood levels of branched-chain ot-ketoacids which inhibit the kinase and activate the complex. The findings that et-chloroisocaproate and physiological concentrations of branched-chain ot-ketoacids can activate the complex in hepatocytes isolated from low-protein-fed rats (34, 35) provide additional support for this mechanism (35, 36).
MATERIALS AND METHODS Source of materials. Enzymes and biochemicals were from Sigma Chemical Company. ot-Chloroisocaproate was obtained from Dr. Ronald Simpson of Sandoz, Inc. Radiolabeled compounds were from DuPont-New England Nuclear, molecular biology reagents from Bethesda Research Laboratories, and protein sequencing reagents from Applied Biosystems, Inc. Sequenase T M was from United States Biochemical Corp.
Analytical methods. Branched-chain et-ketoacids were determined enzymatically with purified branched-chain ot-ketoacid dehydrogenase (37). The dideoxy chain termination method (38) and the Sequenase T M method were used for D N A sequence analysis. Isolation and incubation of hepatocytes. Hepatocytes were isolated from male Wistar rats (150-200 g) given free access to a low-protein (8%) diet (32) by the procedure of Berry and Friend (39) with modifications described previously (40). Hepatocytes were incubated in Krebs-Henseleit buffer supplemented with 2.5% bovine serum albumin under an atmosphere of 95% 02, 5% CO 2 in flasks sealed with rubber serum caps fitted with hanging center wells. Incubations were carried out for 45 minin a shaking water bath maintained at 37°C. Isolation of enzymes. The branched-chain a-ketoacid dehydrogenase complex was purified from rat liver by a phenyl-Sepharose method (41), 3-hydroxyisobutyrate dehydrogenase (EC 1.1.1.31) from rabbit liver (42), and the methylmalonate-semialdehyde dehydrogenase (EC 1.2.1.27) from rat liver (43). Extraction and assay of branched-chain ot-ketoacid dehydrogenase complex and its kinase. Extraction of the complex and its associated
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kinase from freeze-clamped tissue and spectrophotometric assay of enzyme activity were carried out as described by Goodwin et al. (44). Briefly, the frozen tissue was pulverized under liquid N 2 and extracted with a buffer containing Triton X-100, KC1, EDTA, dithiothreitol, protease inhibitors, and a-chloroisocaproate. The suspension was homogenized and clarified by centrifugation. The complex was precipitated with 9% polyethylene glycol. The precipitates were resuspended in buffer and enzyme activity determined spectrophotometrically by the rate of N A D H production from saturating concentrations of substrates and cofactors. The active form and total activity of the complex were determined before and after incubation with a broadspecificity phosphatase, respectively. For enzyme activity measurements with fibroblasts, the cells were collected by trypsinization and treated with oL-chloroisocaproate to activate the complex. Total activity was determined radiochemically using [1-14C]ketoisovaleric acid as substrate (45). For the estimation of kinase activity, homogenates of freeze-clamped tissues were prepared as described above except that o~-chloroisocaproate and serum were omitted from the extraction buffer. The complex was completely activated by dephosphorylation with phosphatase and then precipitated again with 9% polyethylene glycol. The precipitate was resuspended and assayed for kinase activity by determination of the first-order rate constant for ATP-dependent inactivation of dehydrogenase activity at 15°C. Cloning of cDNAs for human and rat liver branched-chain ot-ketoacid dehydrogenase Elot and rat liver 3-hydroxyisobutyrate dehydrogenase. Polyclonal antibodies specific for the E1 component of the complex were used to screen a rat liver hgtll cDNA library (Clontech Laboratories, Inc.) as described previously (46). The clone containing the cDNA insert encoding the E l a subunit was selected with oligonucleotide probes designed from known peptide sequences of Elet. A cDNA for human Elot was obtained by screening a human liver hgtll cDNA library (Dr. Savio Woo, Houston, TX) using the cDNA for rat liver Elo~ as probe. A cDNA clone encoding 3-hydroxyisobutyrate dehydrogenase was isolated by screening the Clontech rat liver cDNA library with a degenerate 17-base oligonucleotide probe corresponding to a portion of the N-terminal amino acid sequence of the enzyme. Northern and Western blot analysis. RNA was isolated by the guanidinium isothiocyanate method (47) and analyzed by electrophoresis and Northern blotting. The blots were probed with cDNAs under conditions described previously (45, 46). Proteins of various extracts and preparations were electrophoresed by SDS-polyacrylamide gel electrophoresis. The proteins were electroblotted to polyvinylidene difluoride membranes and analyzed
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immunochemically using polyclonal antibodies raised against the enzyme or enzyme subunit.
Polymerase chain reaction and allele-specific oligonucleotide hybridization. A first-strand cDNA, generated by reverse transcription of total R N A extracted from tissue or fibroblasts, was subjected to 30-40 cycles of enzymatic amplification by means of thermostable Taq DNA polymerase. Five sets of sense/antisense oligonucleotides used for the amplification were designed from known human cDNA sequences (45). The amplified cDNAs were subcloned into M13, and four independent clones were sequenced for each amplified region. In order to confirm the sequence analysis findings, amplified cDNAs and genomic DNAs were blotted to membranes and subjected to allele-specific oligonucleotide hybridization. The probe used for the normal was GAGCACTACCCACTG; for the mutant GAGCACAACCCACTG.
RESULTS AND DISCUSSION
Regulation of the Activity State of the Branched-Chain ot-Ketoacid Dehydrogenase Complex in Isolated Hepatocytes Hepatocytes isolated from rats fed a low-protein diet provide a good model system for studying factors that regulate flux through the branched-chain ot-ketoacid dehydrogenase complex (35, 36, 48). The enzyme is partially inactivated by phosphorylation in such cells, but can be readily activated by incubation of the hepatocytes with et-chloroisocaproate, an inhibitor of branched-chain a-ketoacid dehydrogenase kinase (Table 1). Flux through the complex parallels the activity state of the complex following the addition of a number of compounds to the incubation medium (Table 1), which is consistent with a rate-limiting role of the complex in branched-chain et-ketoacid catabolism, ot-Ketoisocaproate also stimulates flux and activates the complex, again presumably because this compound, produced by transamination of leucine, is a potent inhibitor o r e the kinase, ot-Ketoisocaproate is effective as a stimulator of flux at concentrations known to occur in blood plasma, making it likely that inhibition of kinase activity by this compound is of physiological importance. In contrast, the ketoacids originating from isoleucine and valine are much less potent inhibitors of the kinase, and as would be expected, these compounds produce much less increase in flux and activation of the complex. 13-Hydroxybutyrate added at high substrate concentrations inhibits flux, and also decreases the activity state of the complex. Kinase inhibitors are partially effective in preventing the inhibition caused by
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TABLE 1. EFFECTS OF a-CHLOROISOCAPROATE,/LHYDROXYBUTYRATE, BRANCHED-CHAIN a-KETOACIDS, AND FLUORIDE ON a-KETOISOVALERATE DECARBOXYLATION AND THE ACTIVITY OF THE BRANCHED-CHAIN a-KETOACID DEHYDROGENASE COMPLEX IN ISOLATED HEPATOCYTES FROM LOW-PROTEIN-FED RATS*
Additions (mM) None t~-Chloroisocaproate (0.1) a-Ketoisocaproate (0.1) ctKeto-/~-methylvalerate (0.1) ct-Ketovalerate (0.1) Sodium fluoride (50) a-Chloroisocaproate + sodium fluoride /%Hydroxybutyrate (20) /3-Hydroxybutyrate + a-chloroisocaproate
Rate of a-ketoisovalerate decarboxylation
Activity of branched-chain ketoacid complex
(nmol/min/g wet wt) 105_+ 16 219 _+ 15 197 _+ 18 142 + 21 123 _+ 21 43_+ 6
(nmol/min/g wet wt) 110_+30 (21) 520 _+ 30 (100) 260 _+ 30 (48) 160 _+ 30 (30) 120 _+25 (23) 28_+11 (5)
93+25 42 _+ 12
58_+15 (11) 18 _+ 11 (3)
187 + 22
400 _+50 (76)
*Hepatocytes isolated from low-protein (8%) fed rats were preincubated 15 min with additions indicated, t~-Keto [1-~4C]isovalerate (0.2 mM) was added and the incubations continued for another 15 min before termination with acid and '4CO2 collection. Activity of branched-chain a-ketoacid dehydrogenase complex was determined after a total of 20 min of incubation. The numbers in parentheses corresponds to the percentage of the value obtained with hepatocytes with only a-chloroisocaproate. Values are means + SE for 4 to 6 hepatocyte preparations. Reproduced with permission from data published in Han et al. (36).
t3-hydroxybutyrate. It is attractive to propose that ~-hydroxybutyrate inactivation of the complex involves an increase in the mitochondrial NADH/NAD + ratio, but no effect of NADH nor NAD + has been found on branched-chain ot-ketoacid dehydrogenase kinase activity (18). Sodium fluoride also results in decreased flux and inactivation of the complex. It is not known whether this is an indirect consequence of the numerous effects of fluoride on metabolic processes or a direct consequence of its known potency as an inhibitor of the branched-chain ot-ketoacid dehydrogenase phosphatase.
Assay for the In Vivo Activity State of the Branched-Chain ~t-Ketoacid Dehydrogenase Complex Freeze-clamping tissue at liquid-N2 temperature has been used by this laboratory to preserve the in vivo activity state of the branched-chain ot-ketoacid dehydrogenase complex (32, 44). The freeze-clamped tissue is extracted with a buffer containing Triton X-100 to solubilize the complex, several protease inhibitors to control proteolysis, ot-chloroisocaproate to inhibit the kinase, and EDTA to inhibit endogenous phosphatases. It has
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not been found necessary to add fluoride to the extraction medium to protect against phosphatase activity during extraction and assay of the enzyme from fed, starved, and low-protein-fed rats (48). For example, activity of the enzyme extracted and assayed without fluoride was 1.47 + 0.08 units/g wet wt; activity extracted and assayed with fluoride (50 mM and 25 mM, respectively) was 1.42 + 0.09 units/g wet wt (mean _ SEM; n = 4 chow-fed rats). These results indicate that EDTA and low temperature are sufficient to inhibit phosphatase activity during extraction of the tissue. This finding was confirmed by adding 32p-enzyme to the tissue prior to extraction and assay of the complex. Only a very small fraction of the added radioactivity was released as 32pi during the procedure. As another check, we added purified enzymes, in either the inactive, phosphorylated or the active, nonphosphorylated forms, to frozen liver powders before extraction. Good recoveries were obtained (49), proving that the method works well for extraction and recovery of both forms of the enzyme. Buse and colleagues (30, 50) introduced the novel step of precipitation of the complex from crude extracts with polyethylene glycol prior to assay. This greatly concentrates the enzyme, allows spectrophotometric assay of the liver enzyme, and has been incorporated into our procedure in recent studies (44).
Regulation of Hepatic Branched-Chain a-Ketoacid Dehydrogenase Complex In Vivo Previous work from this laboratory (9, 32) and others (26-28) has established that protein starvation dramatically decreases the activity state of the branched-chain ot-ketoacid dehydrogenase complex. Actual starvation for calories, however, does not result in inactivation of the complex (32); indeed, starvation of animals fed a low-protein diet results in activation of the hepatic enzyme, presumably because of increased proteolysis and the need for glucose synthesis from amino acids in this state. Induction of diabetes by means of streptozotocin produces effects similar to those of starvation, i.e. no effect in the well-fed rat (because the enzyme is already completely activated), but activation of the enzyme in the low-protein-fed animal (51; R. Gibson and R. A. Harris, unpublished studies). These findings presumably reflect the decreased protein synthesis and increased protein degradation characteristic of the diabetic state. This hypothesis was tested by an experiment in which protein synthesis was blocked in vivo by the injection of cycloheximide (P. Dexter and R. A. Harris, unpublished data). Rats starved for protein were chosen to establish an experimental condition in which branched-chain amino acids are conserved for protein synthesis by the inactivation of the hepatic complex by phosphorylation. Inhibition of protein synthesis by cycloheximide in these
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animals resulted in a large increase in plasma branched-chain ot-ketoacids (from 16 to 39/~M) and, presumably because these ketoacids are inhibitors of the kinase, an increase in the activity state of the hepatic branched-chain ot-ketoacid dehydrogenase complex (from 23 to 74% of the enzyme in the active form). This result, along with the effects of starvation and diabetes, illustrate that the regulation of the branched-chain ot-ketoacid dehydrogenase complex is tied to the need to dispose of branched-chain amino acids available in excess of that required for protein synthesis. Recent work has concentrated on the mechanisms that could explain the alteration in activity state of the complex in different nutritional states. Two types of regulation of the branched-chain oL-ketoacid dehydrogenase kinase have now been recognized. First, experiments with purified enzyme (17, 18) and isolated hepatocytes (36) indicate that the branched chain et-ketoacids are inhibitors of the kinase. Therefore, the ketoacids can stimulate their degradation since the phosphatase can act unopposed to activate the complex. Secondly, adaptive changes in the total activity of the kinase have been found to occur that may represent yet another mechanism of importance in regulation of the phosphorylation state of the complex (52, 53). In a recent study from this laboratory, starvation of rats for protein by placing them on an 8% protein diet for 2 weeks resulted in a 3.4-fold increase in total hepatic branched-chain et-ketoacid dehydrogenase kinase, a 30% decrease in blood branched-chain ot-ketoacids levels, and a 45% decrease in hepatic total branched-chain et-ketoacid dehydrogenase activity (Table 2). The increase in kinase activity and the decrease in branched-chain (xketoacid levels may act in a cooperative manner to effect inactivation of the
T A B L E 2. B L O O D B R A N C H E D - C H A I N a - K E T O A C I D LEVELS A N D LIVER B R A N C H E D - C H A I N a - K E T O A C I D D E H Y D R O G E N A S E K I N A S E ACTIVITY, I M M U N O R E A C T I V E El, A N D B R A N C H E D - C H A I N a - K E T O A C I D D E H Y D R O G E N A S E C O M P L E X ACTIVITY STATE IN V A R I O U S N U T R I T I O N A L STATES*
Nutritional state
Chow-feed 48-hr starved Low-protein fed Low-protein fed then starved
Branched-chain a-ketoacids
Kinase activity
lmmunoreactive E1
(vl/mg
Complex activity state
(/./,M)
(min -l)
23 + 1 32 + 4 17 + 2
0.25 + 0.05 0.10 + 0.01 0.86 + 0.05
wet wt) 2.44 + 0.32 2.44 + 0.29 1.35 + 0.02
(%) 96 + 3 102 + 3 13 + 3
37 + 4
0.56 + 0.09
not determined
89 + 5
*Rats were either fed a chow diet or a low-protein (8%) diet for 2 weeks prior to the experiment. Immunoreactive E 1 is expressed as the a m o u n t of E 1 antiserum required to completely inactivate dehydrogenase activity of the complex. Unpublished data o f G . W. Goodwin, Y. S h i m o m u r a a n d R. A. Harris.
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complex in this nutritional state. Subsequent starvation of low-proteinfed rats reactivates the complex (32, Table 2) and normalizes the branched-chain et-ketoacid levels, while the kinase activity remains the same as before starvation, suggesting that the mechanism of reactivation is inhibition of the kinase by the branched-chain c~-ketoacids with little contribution of a change in total kinase activity. Nevertheless, the dose response of dietary protein intake on the activity state of the complex and the activity of the kinase over the normal dietary range of protein (Fig. 1) suggests that the activity of the kinase is major determinant of the activity state of the complex. It would appear from these findings and those of others (52, 54) that short-term regulation of the activity state of the complex can be brought about by changes in branched-chain ot-ketoacid levels whereas long-term regulation of the complex is effected by changes in the activity of the branched-chain et-ketoacid dehydrogenase kinase. Exactly how adaptive changes in kinase activity occur remains to be determined. Likewise, for want of an assay, it has not been determined whether adaptive changes
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FIG. 1. Dose response of dietary protein intake on the activity state of the hepatic branched-chain a-ketoacid dehydrogenase complex (O) and the activity of branched-chain a-ketoacid dehydrogenase kinase (tl)) in liver. Rats were maintained ad libitum on diets of the indicated protein concentration for 2 weeks. Livers were freeze-clamped at liquid-N2 temperature no more than 10 sec after cervical dislocation. The activity state of the complex and the activity of the kinase were determined as described under Materials and Methods. Values given at the means + SE for 5 animals at each dietary protein level. Data taken from unpublished work of G. W. Goodwin, Y. Shimomura, and R. A. Harris.
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also occur in the activity of the branched-chain ot-ketoacid dehydrogenase phosphatase. Monovalent Cations and Inorganic Phosphate Alter Branched-chain otKetoacid Dehydrogenase Kinase Activity and Inhibitor Sensitivity We have found that potassium ion protects the branched-chain ot-ketoacid dehydrogenase complex against inactivation by thermal denaturation and protease digestion (55). Thiamine pyrophosphate stabilization of the complex (56) is also dependent on the presence of potassium ion (55). Kinase activity as measured by inactivation of the complex was maximized by 100 mM potassium ion. Several salts were found to increase the efficiency of inactivation of the complex by phosphorylation, i.e., decreased the degree of enzyme phosphorylation required to cause inactivation of the complex (Table 3). The effectiveness and efficacy of kinase inhibitors (o~-chloroisocaproate) were enhanced by the presence of monovalent cations and inorganic phosphate (Fig. 2). These findings suggest that monovalent cations and phosphate cause structural changes in the complex that alter its susceptibility to phosphorylation and responsiveness to activators and inhibitors. Since the complex exhibits this sensitivity to experimental conditions, attempts to identify physiologically important effectors may have underestimated the effectiveness of various compounds. This may be of considerable importance because no explanation is available for the observed effects of several compounds on the activity state of the complex in isolated hepatocytes (35, 36). Two phosphorylation sites, separated by 9 amino acids, are present in the Elet subunit. Phosphorylation of site 1 correlates best with inhibition TABLE 3. EFFECTS OF VARIOUS SALTS ON BRANCHED-CHAIN a-KETOACID DEHYDROGENASE KINASE ACTIVITY* Salt added (mM) None KCI (100) K2Pi (50) LiCI (100) NaCI (100)
Kinase activity By complex By phosphate inactivation incorporation (min -I) 0.064 0.107 0.119 0.064 0.064
(nmol/30 min/mg) 7.4 6.4 6.6 5.2 5.4
Phosphorylation efficiency × 102 0.86 1.67 1.80 1.23 1.19
*Kinase activity was assayed by inactivation of dehydrogenase activity of the complex and by 32p incorporation from radioactive ATP into E 1t~ protein. Phosphorylation efficiency is the rate of complex inactivation divided by the extent of phosphorylation at 30 rain. Data taken from Shimomura et al. (55).
BRANCHED-CHAINa-KETOACIDDEHYDROGENASE
I
~W4),'''~ -
255
50mMK-pho,phate
80
i 60 4O
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FIG. 2. Inhibition of branched-chain ee-ketoacid dehydrogenase kinase activity by ot-chloroisocaproate. Activityof the complexwas assayed 10 and 20 min after initiationof the incubation.First-orderrate constants,calculatedfromthe ratesof lossof complexactivity, were used to calculate the percentage inhibition by c~-chloroisocaproate. Reproduced with permissionfrom Shimomuraet al. (55). of enzyme activity (21, 22). However, it has not been possible to conclude that phosphorylation of site 2 is completely silent. Both sites are readily phosphorylated in vivo (57) and in vitro (21, 22). Inhibitors of the kinase are much more effective against phosphorylation of site 2 than site 1 (22). Experimental conditions (ionic strength, pH, cation composition of the buffer) profoundly affect site 2 but not site I phosphorylation (M. J. Kuntz, Y. Shimomura and R. A. Harris, unpublished results), and this provides a mechanism for the increased phosphorylation efficiency found for various salts (Table 2). These findings raise the question of whether two different kinases might be associated with the complex. Isolation and characterization of the branched-chain ot-ketoacid dehydrogenase kinase(s) would resolve this issue.
Molecular Cloning of cDNAs for the Elet Subunit of Rat and Human Liver Branched-chain et-Ketoacid Dehydrogenase Complex A cDNA encoding the rat branched-chain ot-ketoacid dehydrogenase El~t subunit was obtained by screening a liver kgt11 expression library with a polyclonal antibody against the E1 complex (57). The 1.7 kb cDNA contains an open reading frame of 1,323 base pairs which includes the code for 24 amino acid residues surrounding phosphorylation sites 1 and 2 of the bovine kidney (21) and rabbit heart (22) enzymes plus the N-terminal sequence of the rat liver enzyme (46). A 1,552 base pair human BCKDH Elot subunit cDNA was isolated from a human liver cDNA library by using the rat cDNA as a probe (58) and a cDNA fragment encoding the N-terminus
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of the human enzyme was later obtained by amplification of human cDNA by polymerase chain reaction. The predicted human polypeptide shows 96% homology with the rat EloL subunit. The 117 amino acid residues surrounding the phosphorylation sites are completely conserved between human and rat, indicating the importance of this region in the function of the subunit. Considerable amino acid sequence similarity is observed with pyruvate dehydrogenase EloL, particularly in the region of the phosphorylation sites of these proteins (49% similarity within the amino acid region of 252-327 when conservative substitutions are allowed).
Elucidation of a Molecular Basis for Maple Syrup Urine Disease Fibroblasts from four maple syrup urine disease patients and one animal model of the disease are currently being grown in culture in the laboratory. Northern blots of normal human liver and fibroblast R N A demonstrate a single 1.8 kb mRNA band when probed with EI~ eDNA. A method has been developed for rapid sequence analysis of mutant EI~ mRNAs. The EI~ mRNA from liver or fibroblasts is amplified by synthesizing a set of cDNAs primed with specific oligonucleotides and then performing the polymerase chain reaction with thermostable Taq DNA polymerase. The resulting amplified DNAs are cloned into M13 for sequencing. The etiology of maple syrup urine disease in one patient with the classic form of the disease has been studied (45). A transversion of T to A that changes a tyrosine to an asparagine at residue 394 of the E l a was found (Fig. 3). Amplification of both mRNA and genomic DNA, in combination with allele-specific oligonucleotide hybridization, demonstrated that the father was heterozygous for this mutant allele (Fig. 4). The mother was homozygous for the allele encoding the normal Tyr-394, but expressed only about half of the normal level of mRNA. The patient is a compound heterozygote, inheriting an allele from the father encoding an abnormal EI~, and an allele from the mother containing a cis-aeting defect in regulation which abolishes the expression of one of the EI~ alleles. Maple syrup urine disease occurs in the Polled Hereford breed of cattle in Australia (59). A single base substitution at codon --6 (CAG to TAG) is responsible for the defect in these cattle, the only known animal model of the disease (B. Zhang, P. J. Healy, Y. Zhao, D. W. Crabb and R. A. Harris, J. Biol. chem., in press (1990)). This mutation introduces a stop codon in the leader peptide of the EI~ subunit, resulting in premature termination of translation and apparently destabilization of the mRNA. Some patients with maple syrup urine disease will respond to thiamine administration with a reduction in blood branched-chain ~-ketoaeids (60). Thiamine administration may increase tissue levels of thiamine pyrophosphate which may compensate for defective binding of this
INTRODUCTION
With the exception of the valine pathway (l), the enzyme-catalyzed reactions for the oxidation of branched-chain amino acids are well established. A high-capacity system for branched-chain amino acid catabolism is needed to prevent severe toxic effects of these amino acids and their metabolites that are characteristic of maple syrup urine disease. Nevertheless, catabolism of branched-chain amino acids must be under tight control, particularly when dietary protein intake is restricted, to assure availability of these essential amino acids for protein synthesis. Various physiological conditions (e.g. starvation, diabetes, trauma) influence BCAA metabolism, attesting to the importance of the regulatory mechanisms for their catabolism. There is much interest in mechanisms that regulate branched-chain amino acid levels because leucine has been shown to inhibit protein degradation (2), stimulate protein synthesis (3), and promote insulin release (4, 5). Although controversial, there is also interest in a possible role of branched-chain amino acids in the pathogenesis and treatment of hepatic encephalopathy (6). Regulation of branched-chain amino acid catabolism is achieved in large part at the branched-chain ol-ketoacid dehydrogenase complex (7-9), the enzyme responsible for oxidative decarboxylation of the a-ketoacids derived by transamination of branched-chain amino acids. This multienzyme complex, composed of an a-ketoacid decarboxylase (El, EC 1.2.4.4), a transacylase (E2, no EC number), and dihydrolipoamide reductase (E3, EC 1.8.1.4), is subject to regulation by covalent modification (l&12). The kinase (no EC number) which phosphorylates the Ela subunit and 245
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inactivates the decarboxylase is also tightly associated with the complex. E1 is composed of two different subunits in an a2132 structure (13); there are 24 of each of the E1 subunits and 24 E2 subunits in the complex (14, 15). The pioneering work of the laboratories of Reed (15) and Danner (16) in purification of the complex opened the door for the study of its regulation. The kinase is inhibited by branched-chain et-ketoacids (17, 18), occhloroisocaproate (19), and dichloroacetate (20). Phosphorylation occurs on two serine residues of the Elot subunit, with site 1 (Ser 293) probably more important than site 2 (Ser 303) in modifying enzyme activity (21, 22). The kinase has neither been purified nor resolved from the complex. In contrast, the kinase (EC2.7.1.99) responsible for phosphorylation of the related pyruvate dehydrogenase complex (EC 1.2.4.1 + EC 2.3.1.12 + EC 1.8.1.4) has been isolated and partially characterized. It has an ot13 structure with subunit molecular weights of 48,000 and 45,000 (23). A protein of 45,000 daltons which may correspond to a kinase subunit has been observed by SDS-polyacrylamide gel electrophoresis with some preparations of the branched-chain otketoacid dehydrogenase complex (12). The branched-chain a-ketoacid dehydrogenase phosphatase (no EC number), also known to be distinct from pyruvate dehydrogenase phosphatase (EC 3.1.3.43), has an apparent M r of 460,000 with a catalytic subunit of 33,000 (24, 25). Regulation of the activity state of the branched-chain ot-ketoacid dehydrogenase in various physiological states has been studied in the laboratories (7-9, 26-31). As summarized below, there is basic agreement with respect to the activity state of the hepatic enzyme in various nutritional states. Restriction of protein intake by feeding diets low in protein results in activation of hepatic branched-chain ot-ketoacid dehydrogenase complex (9, 32), thereby conserving branched-chain amino acids for protein synthesis during periods of protein starvation. Protein intake beyond that needed for maximum growth maintains branched-chain et-ketoacid dehydrogenase complex in the active state, thereby providing a mechanism for disposal of branched-chain amino acids in excess of that required for protein synthesis. The original experiments (33, 34) demonstrating decreased capacity of the liver in rats fed a low-protein diet to oxidize branched-chain keto acids are now readily explained by covalent modification of the branched-chain aketoacid dehydrogenase complex. Starvation of rats previously maintained on chow diet causes no significant change in the activity state of hepatic branched-chain a-ketoacid dehydrogenase complex (9, 32). Starvation of rats previously fed a low-protein (8%) diet, in which the enzyme is initially in a low-activity state, results in activation (9, 32). Inactivation of the complex observed in the protein-deficient state may be due to less
BRANCHED-CHAINot-KETOACIDDEHYDROGENASE
247
inhibition of the kinase because of reduced plasma and tissue concentrations of branched-chain ot-ketoacids. No change in activity state is observed upon starvation of chow-fed rats because excess branched-chain ketoacids are supplied to the liver in both cases, i.e. by digestion of dietary protein in the fed state and by endogenous proteolysis in the starved state. Starvation of rats fed low-protein diets promotes endogenous proteolysis, giving rise to increased blood levels of branched-chain ot-ketoacids which inhibit the kinase and activate the complex. The findings that et-chloroisocaproate and physiological concentrations of branched-chain ot-ketoacids can activate the complex in hepatocytes isolated from low-protein-fed rats (34, 35) provide additional support for this mechanism (35, 36).
MATERIALS AND METHODS Source of materials. Enzymes and biochemicals were from Sigma Chemical Company. ot-Chloroisocaproate was obtained from Dr. Ronald Simpson of Sandoz, Inc. Radiolabeled compounds were from DuPont-New England Nuclear, molecular biology reagents from Bethesda Research Laboratories, and protein sequencing reagents from Applied Biosystems, Inc. Sequenase T M was from United States Biochemical Corp.
Analytical methods. Branched-chain et-ketoacids were determined enzymatically with purified branched-chain ot-ketoacid dehydrogenase (37). The dideoxy chain termination method (38) and the Sequenase T M method were used for D N A sequence analysis. Isolation and incubation of hepatocytes. Hepatocytes were isolated from male Wistar rats (150-200 g) given free access to a low-protein (8%) diet (32) by the procedure of Berry and Friend (39) with modifications described previously (40). Hepatocytes were incubated in Krebs-Henseleit buffer supplemented with 2.5% bovine serum albumin under an atmosphere of 95% 02, 5% CO 2 in flasks sealed with rubber serum caps fitted with hanging center wells. Incubations were carried out for 45 minin a shaking water bath maintained at 37°C. Isolation of enzymes. The branched-chain a-ketoacid dehydrogenase complex was purified from rat liver by a phenyl-Sepharose method (41), 3-hydroxyisobutyrate dehydrogenase (EC 1.1.1.31) from rabbit liver (42), and the methylmalonate-semialdehyde dehydrogenase (EC 1.2.1.27) from rat liver (43). Extraction and assay of branched-chain ot-ketoacid dehydrogenase complex and its kinase. Extraction of the complex and its associated
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kinase from freeze-clamped tissue and spectrophotometric assay of enzyme activity were carried out as described by Goodwin et al. (44). Briefly, the frozen tissue was pulverized under liquid N 2 and extracted with a buffer containing Triton X-100, KC1, EDTA, dithiothreitol, protease inhibitors, and a-chloroisocaproate. The suspension was homogenized and clarified by centrifugation. The complex was precipitated with 9% polyethylene glycol. The precipitates were resuspended in buffer and enzyme activity determined spectrophotometrically by the rate of N A D H production from saturating concentrations of substrates and cofactors. The active form and total activity of the complex were determined before and after incubation with a broadspecificity phosphatase, respectively. For enzyme activity measurements with fibroblasts, the cells were collected by trypsinization and treated with oL-chloroisocaproate to activate the complex. Total activity was determined radiochemically using [1-14C]ketoisovaleric acid as substrate (45). For the estimation of kinase activity, homogenates of freeze-clamped tissues were prepared as described above except that o~-chloroisocaproate and serum were omitted from the extraction buffer. The complex was completely activated by dephosphorylation with phosphatase and then precipitated again with 9% polyethylene glycol. The precipitate was resuspended and assayed for kinase activity by determination of the first-order rate constant for ATP-dependent inactivation of dehydrogenase activity at 15°C. Cloning of cDNAs for human and rat liver branched-chain ot-ketoacid dehydrogenase Elot and rat liver 3-hydroxyisobutyrate dehydrogenase. Polyclonal antibodies specific for the E1 component of the complex were used to screen a rat liver hgtll cDNA library (Clontech Laboratories, Inc.) as described previously (46). The clone containing the cDNA insert encoding the E l a subunit was selected with oligonucleotide probes designed from known peptide sequences of Elet. A cDNA for human Elot was obtained by screening a human liver hgtll cDNA library (Dr. Savio Woo, Houston, TX) using the cDNA for rat liver Elo~ as probe. A cDNA clone encoding 3-hydroxyisobutyrate dehydrogenase was isolated by screening the Clontech rat liver cDNA library with a degenerate 17-base oligonucleotide probe corresponding to a portion of the N-terminal amino acid sequence of the enzyme. Northern and Western blot analysis. RNA was isolated by the guanidinium isothiocyanate method (47) and analyzed by electrophoresis and Northern blotting. The blots were probed with cDNAs under conditions described previously (45, 46). Proteins of various extracts and preparations were electrophoresed by SDS-polyacrylamide gel electrophoresis. The proteins were electroblotted to polyvinylidene difluoride membranes and analyzed
BRANCHED-CHAIN~t-KETOACIDDEHYDROGENASE
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immunochemically using polyclonal antibodies raised against the enzyme or enzyme subunit.
Polymerase chain reaction and allele-specific oligonucleotide hybridization. A first-strand cDNA, generated by reverse transcription of total R N A extracted from tissue or fibroblasts, was subjected to 30-40 cycles of enzymatic amplification by means of thermostable Taq DNA polymerase. Five sets of sense/antisense oligonucleotides used for the amplification were designed from known human cDNA sequences (45). The amplified cDNAs were subcloned into M13, and four independent clones were sequenced for each amplified region. In order to confirm the sequence analysis findings, amplified cDNAs and genomic DNAs were blotted to membranes and subjected to allele-specific oligonucleotide hybridization. The probe used for the normal was GAGCACTACCCACTG; for the mutant GAGCACAACCCACTG.
RESULTS AND DISCUSSION
Regulation of the Activity State of the Branched-Chain ot-Ketoacid Dehydrogenase Complex in Isolated Hepatocytes Hepatocytes isolated from rats fed a low-protein diet provide a good model system for studying factors that regulate flux through the branched-chain ot-ketoacid dehydrogenase complex (35, 36, 48). The enzyme is partially inactivated by phosphorylation in such cells, but can be readily activated by incubation of the hepatocytes with et-chloroisocaproate, an inhibitor of branched-chain a-ketoacid dehydrogenase kinase (Table 1). Flux through the complex parallels the activity state of the complex following the addition of a number of compounds to the incubation medium (Table 1), which is consistent with a rate-limiting role of the complex in branched-chain et-ketoacid catabolism, ot-Ketoisocaproate also stimulates flux and activates the complex, again presumably because this compound, produced by transamination of leucine, is a potent inhibitor o r e the kinase, ot-Ketoisocaproate is effective as a stimulator of flux at concentrations known to occur in blood plasma, making it likely that inhibition of kinase activity by this compound is of physiological importance. In contrast, the ketoacids originating from isoleucine and valine are much less potent inhibitors of the kinase, and as would be expected, these compounds produce much less increase in flux and activation of the complex. 13-Hydroxybutyrate added at high substrate concentrations inhibits flux, and also decreases the activity state of the complex. Kinase inhibitors are partially effective in preventing the inhibition caused by
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TABLE 1. EFFECTS OF a-CHLOROISOCAPROATE,/LHYDROXYBUTYRATE, BRANCHED-CHAIN a-KETOACIDS, AND FLUORIDE ON a-KETOISOVALERATE DECARBOXYLATION AND THE ACTIVITY OF THE BRANCHED-CHAIN a-KETOACID DEHYDROGENASE COMPLEX IN ISOLATED HEPATOCYTES FROM LOW-PROTEIN-FED RATS*
Additions (mM) None t~-Chloroisocaproate (0.1) a-Ketoisocaproate (0.1) ctKeto-/~-methylvalerate (0.1) ct-Ketovalerate (0.1) Sodium fluoride (50) a-Chloroisocaproate + sodium fluoride /%Hydroxybutyrate (20) /3-Hydroxybutyrate + a-chloroisocaproate
Rate of a-ketoisovalerate decarboxylation
Activity of branched-chain ketoacid complex
(nmol/min/g wet wt) 105_+ 16 219 _+ 15 197 _+ 18 142 + 21 123 _+ 21 43_+ 6
(nmol/min/g wet wt) 110_+30 (21) 520 _+ 30 (100) 260 _+ 30 (48) 160 _+ 30 (30) 120 _+25 (23) 28_+11 (5)
93+25 42 _+ 12
58_+15 (11) 18 _+ 11 (3)
187 + 22
400 _+50 (76)
*Hepatocytes isolated from low-protein (8%) fed rats were preincubated 15 min with additions indicated, t~-Keto [1-~4C]isovalerate (0.2 mM) was added and the incubations continued for another 15 min before termination with acid and '4CO2 collection. Activity of branched-chain a-ketoacid dehydrogenase complex was determined after a total of 20 min of incubation. The numbers in parentheses corresponds to the percentage of the value obtained with hepatocytes with only a-chloroisocaproate. Values are means + SE for 4 to 6 hepatocyte preparations. Reproduced with permission from data published in Han et al. (36).
t3-hydroxybutyrate. It is attractive to propose that ~-hydroxybutyrate inactivation of the complex involves an increase in the mitochondrial NADH/NAD + ratio, but no effect of NADH nor NAD + has been found on branched-chain ot-ketoacid dehydrogenase kinase activity (18). Sodium fluoride also results in decreased flux and inactivation of the complex. It is not known whether this is an indirect consequence of the numerous effects of fluoride on metabolic processes or a direct consequence of its known potency as an inhibitor of the branched-chain ot-ketoacid dehydrogenase phosphatase.
Assay for the In Vivo Activity State of the Branched-Chain ~t-Ketoacid Dehydrogenase Complex Freeze-clamping tissue at liquid-N2 temperature has been used by this laboratory to preserve the in vivo activity state of the branched-chain ot-ketoacid dehydrogenase complex (32, 44). The freeze-clamped tissue is extracted with a buffer containing Triton X-100 to solubilize the complex, several protease inhibitors to control proteolysis, ot-chloroisocaproate to inhibit the kinase, and EDTA to inhibit endogenous phosphatases. It has
BRANCHED-CHAIN~t-KETOACIDDEHYDROGENASE
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not been found necessary to add fluoride to the extraction medium to protect against phosphatase activity during extraction and assay of the enzyme from fed, starved, and low-protein-fed rats (48). For example, activity of the enzyme extracted and assayed without fluoride was 1.47 + 0.08 units/g wet wt; activity extracted and assayed with fluoride (50 mM and 25 mM, respectively) was 1.42 + 0.09 units/g wet wt (mean _ SEM; n = 4 chow-fed rats). These results indicate that EDTA and low temperature are sufficient to inhibit phosphatase activity during extraction of the tissue. This finding was confirmed by adding 32p-enzyme to the tissue prior to extraction and assay of the complex. Only a very small fraction of the added radioactivity was released as 32pi during the procedure. As another check, we added purified enzymes, in either the inactive, phosphorylated or the active, nonphosphorylated forms, to frozen liver powders before extraction. Good recoveries were obtained (49), proving that the method works well for extraction and recovery of both forms of the enzyme. Buse and colleagues (30, 50) introduced the novel step of precipitation of the complex from crude extracts with polyethylene glycol prior to assay. This greatly concentrates the enzyme, allows spectrophotometric assay of the liver enzyme, and has been incorporated into our procedure in recent studies (44).
Regulation of Hepatic Branched-Chain a-Ketoacid Dehydrogenase Complex In Vivo Previous work from this laboratory (9, 32) and others (26-28) has established that protein starvation dramatically decreases the activity state of the branched-chain ot-ketoacid dehydrogenase complex. Actual starvation for calories, however, does not result in inactivation of the complex (32); indeed, starvation of animals fed a low-protein diet results in activation of the hepatic enzyme, presumably because of increased proteolysis and the need for glucose synthesis from amino acids in this state. Induction of diabetes by means of streptozotocin produces effects similar to those of starvation, i.e. no effect in the well-fed rat (because the enzyme is already completely activated), but activation of the enzyme in the low-protein-fed animal (51; R. Gibson and R. A. Harris, unpublished studies). These findings presumably reflect the decreased protein synthesis and increased protein degradation characteristic of the diabetic state. This hypothesis was tested by an experiment in which protein synthesis was blocked in vivo by the injection of cycloheximide (P. Dexter and R. A. Harris, unpublished data). Rats starved for protein were chosen to establish an experimental condition in which branched-chain amino acids are conserved for protein synthesis by the inactivation of the hepatic complex by phosphorylation. Inhibition of protein synthesis by cycloheximide in these
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animals resulted in a large increase in plasma branched-chain ot-ketoacids (from 16 to 39/~M) and, presumably because these ketoacids are inhibitors of the kinase, an increase in the activity state of the hepatic branched-chain ot-ketoacid dehydrogenase complex (from 23 to 74% of the enzyme in the active form). This result, along with the effects of starvation and diabetes, illustrate that the regulation of the branched-chain ot-ketoacid dehydrogenase complex is tied to the need to dispose of branched-chain amino acids available in excess of that required for protein synthesis. Recent work has concentrated on the mechanisms that could explain the alteration in activity state of the complex in different nutritional states. Two types of regulation of the branched-chain oL-ketoacid dehydrogenase kinase have now been recognized. First, experiments with purified enzyme (17, 18) and isolated hepatocytes (36) indicate that the branched chain et-ketoacids are inhibitors of the kinase. Therefore, the ketoacids can stimulate their degradation since the phosphatase can act unopposed to activate the complex. Secondly, adaptive changes in the total activity of the kinase have been found to occur that may represent yet another mechanism of importance in regulation of the phosphorylation state of the complex (52, 53). In a recent study from this laboratory, starvation of rats for protein by placing them on an 8% protein diet for 2 weeks resulted in a 3.4-fold increase in total hepatic branched-chain et-ketoacid dehydrogenase kinase, a 30% decrease in blood branched-chain ot-ketoacids levels, and a 45% decrease in hepatic total branched-chain et-ketoacid dehydrogenase activity (Table 2). The increase in kinase activity and the decrease in branched-chain (xketoacid levels may act in a cooperative manner to effect inactivation of the
T A B L E 2. B L O O D B R A N C H E D - C H A I N a - K E T O A C I D LEVELS A N D LIVER B R A N C H E D - C H A I N a - K E T O A C I D D E H Y D R O G E N A S E K I N A S E ACTIVITY, I M M U N O R E A C T I V E El, A N D B R A N C H E D - C H A I N a - K E T O A C I D D E H Y D R O G E N A S E C O M P L E X ACTIVITY STATE IN V A R I O U S N U T R I T I O N A L STATES*
Nutritional state
Chow-feed 48-hr starved Low-protein fed Low-protein fed then starved
Branched-chain a-ketoacids
Kinase activity
lmmunoreactive E1
(vl/mg
Complex activity state
(/./,M)
(min -l)
23 + 1 32 + 4 17 + 2
0.25 + 0.05 0.10 + 0.01 0.86 + 0.05
wet wt) 2.44 + 0.32 2.44 + 0.29 1.35 + 0.02
(%) 96 + 3 102 + 3 13 + 3
37 + 4
0.56 + 0.09
not determined
89 + 5
*Rats were either fed a chow diet or a low-protein (8%) diet for 2 weeks prior to the experiment. Immunoreactive E 1 is expressed as the a m o u n t of E 1 antiserum required to completely inactivate dehydrogenase activity of the complex. Unpublished data o f G . W. Goodwin, Y. S h i m o m u r a a n d R. A. Harris.
BRANCHED-CHAIN tx-KETOACID DEHYDROGENASE
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complex in this nutritional state. Subsequent starvation of low-proteinfed rats reactivates the complex (32, Table 2) and normalizes the branched-chain et-ketoacid levels, while the kinase activity remains the same as before starvation, suggesting that the mechanism of reactivation is inhibition of the kinase by the branched-chain c~-ketoacids with little contribution of a change in total kinase activity. Nevertheless, the dose response of dietary protein intake on the activity state of the complex and the activity of the kinase over the normal dietary range of protein (Fig. 1) suggests that the activity of the kinase is major determinant of the activity state of the complex. It would appear from these findings and those of others (52, 54) that short-term regulation of the activity state of the complex can be brought about by changes in branched-chain ot-ketoacid levels whereas long-term regulation of the complex is effected by changes in the activity of the branched-chain et-ketoacid dehydrogenase kinase. Exactly how adaptive changes in kinase activity occur remains to be determined. Likewise, for want of an assay, it has not been determined whether adaptive changes
ioo
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1O
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!
2.S
2.0
60 ~g
m
40
1.0
~7 v 0.5 U m
I
I
1
I
I
I
0
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20
~0
40
SO
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FIG. 1. Dose response of dietary protein intake on the activity state of the hepatic branched-chain a-ketoacid dehydrogenase complex (O) and the activity of branched-chain a-ketoacid dehydrogenase kinase (tl)) in liver. Rats were maintained ad libitum on diets of the indicated protein concentration for 2 weeks. Livers were freeze-clamped at liquid-N2 temperature no more than 10 sec after cervical dislocation. The activity state of the complex and the activity of the kinase were determined as described under Materials and Methods. Values given at the means + SE for 5 animals at each dietary protein level. Data taken from unpublished work of G. W. Goodwin, Y. Shimomura, and R. A. Harris.
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also occur in the activity of the branched-chain ot-ketoacid dehydrogenase phosphatase. Monovalent Cations and Inorganic Phosphate Alter Branched-chain otKetoacid Dehydrogenase Kinase Activity and Inhibitor Sensitivity We have found that potassium ion protects the branched-chain ot-ketoacid dehydrogenase complex against inactivation by thermal denaturation and protease digestion (55). Thiamine pyrophosphate stabilization of the complex (56) is also dependent on the presence of potassium ion (55). Kinase activity as measured by inactivation of the complex was maximized by 100 mM potassium ion. Several salts were found to increase the efficiency of inactivation of the complex by phosphorylation, i.e., decreased the degree of enzyme phosphorylation required to cause inactivation of the complex (Table 3). The effectiveness and efficacy of kinase inhibitors (o~-chloroisocaproate) were enhanced by the presence of monovalent cations and inorganic phosphate (Fig. 2). These findings suggest that monovalent cations and phosphate cause structural changes in the complex that alter its susceptibility to phosphorylation and responsiveness to activators and inhibitors. Since the complex exhibits this sensitivity to experimental conditions, attempts to identify physiologically important effectors may have underestimated the effectiveness of various compounds. This may be of considerable importance because no explanation is available for the observed effects of several compounds on the activity state of the complex in isolated hepatocytes (35, 36). Two phosphorylation sites, separated by 9 amino acids, are present in the Elet subunit. Phosphorylation of site 1 correlates best with inhibition TABLE 3. EFFECTS OF VARIOUS SALTS ON BRANCHED-CHAIN a-KETOACID DEHYDROGENASE KINASE ACTIVITY* Salt added (mM) None KCI (100) K2Pi (50) LiCI (100) NaCI (100)
Kinase activity By complex By phosphate inactivation incorporation (min -I) 0.064 0.107 0.119 0.064 0.064
(nmol/30 min/mg) 7.4 6.4 6.6 5.2 5.4
Phosphorylation efficiency × 102 0.86 1.67 1.80 1.23 1.19
*Kinase activity was assayed by inactivation of dehydrogenase activity of the complex and by 32p incorporation from radioactive ATP into E 1t~ protein. Phosphorylation efficiency is the rate of complex inactivation divided by the extent of phosphorylation at 30 rain. Data taken from Shimomura et al. (55).
BRANCHED-CHAINa-KETOACIDDEHYDROGENASE
I
~W4),'''~ -
255
50mMK-pho,phate
80
i 60 4O
lOmMKCl
'./--e
5O0 a-Chloroisocaproate (#M)
FIG. 2. Inhibition of branched-chain ee-ketoacid dehydrogenase kinase activity by ot-chloroisocaproate. Activityof the complexwas assayed 10 and 20 min after initiationof the incubation.First-orderrate constants,calculatedfromthe ratesof lossof complexactivity, were used to calculate the percentage inhibition by c~-chloroisocaproate. Reproduced with permissionfrom Shimomuraet al. (55). of enzyme activity (21, 22). However, it has not been possible to conclude that phosphorylation of site 2 is completely silent. Both sites are readily phosphorylated in vivo (57) and in vitro (21, 22). Inhibitors of the kinase are much more effective against phosphorylation of site 2 than site 1 (22). Experimental conditions (ionic strength, pH, cation composition of the buffer) profoundly affect site 2 but not site I phosphorylation (M. J. Kuntz, Y. Shimomura and R. A. Harris, unpublished results), and this provides a mechanism for the increased phosphorylation efficiency found for various salts (Table 2). These findings raise the question of whether two different kinases might be associated with the complex. Isolation and characterization of the branched-chain ot-ketoacid dehydrogenase kinase(s) would resolve this issue.
Molecular Cloning of cDNAs for the Elet Subunit of Rat and Human Liver Branched-chain et-Ketoacid Dehydrogenase Complex A cDNA encoding the rat branched-chain ot-ketoacid dehydrogenase El~t subunit was obtained by screening a liver kgt11 expression library with a polyclonal antibody against the E1 complex (57). The 1.7 kb cDNA contains an open reading frame of 1,323 base pairs which includes the code for 24 amino acid residues surrounding phosphorylation sites 1 and 2 of the bovine kidney (21) and rabbit heart (22) enzymes plus the N-terminal sequence of the rat liver enzyme (46). A 1,552 base pair human BCKDH Elot subunit cDNA was isolated from a human liver cDNA library by using the rat cDNA as a probe (58) and a cDNA fragment encoding the N-terminus
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of the human enzyme was later obtained by amplification of human cDNA by polymerase chain reaction. The predicted human polypeptide shows 96% homology with the rat EloL subunit. The 117 amino acid residues surrounding the phosphorylation sites are completely conserved between human and rat, indicating the importance of this region in the function of the subunit. Considerable amino acid sequence similarity is observed with pyruvate dehydrogenase EloL, particularly in the region of the phosphorylation sites of these proteins (49% similarity within the amino acid region of 252-327 when conservative substitutions are allowed).
Elucidation of a Molecular Basis for Maple Syrup Urine Disease Fibroblasts from four maple syrup urine disease patients and one animal model of the disease are currently being grown in culture in the laboratory. Northern blots of normal human liver and fibroblast R N A demonstrate a single 1.8 kb mRNA band when probed with EI~ eDNA. A method has been developed for rapid sequence analysis of mutant EI~ mRNAs. The EI~ mRNA from liver or fibroblasts is amplified by synthesizing a set of cDNAs primed with specific oligonucleotides and then performing the polymerase chain reaction with thermostable Taq DNA polymerase. The resulting amplified DNAs are cloned into M13 for sequencing. The etiology of maple syrup urine disease in one patient with the classic form of the disease has been studied (45). A transversion of T to A that changes a tyrosine to an asparagine at residue 394 of the E l a was found (Fig. 3). Amplification of both mRNA and genomic DNA, in combination with allele-specific oligonucleotide hybridization, demonstrated that the father was heterozygous for this mutant allele (Fig. 4). The mother was homozygous for the allele encoding the normal Tyr-394, but expressed only about half of the normal level of mRNA. The patient is a compound heterozygote, inheriting an allele from the father encoding an abnormal EI~, and an allele from the mother containing a cis-aeting defect in regulation which abolishes the expression of one of the EI~ alleles. Maple syrup urine disease occurs in the Polled Hereford breed of cattle in Australia (59). A single base substitution at codon --6 (CAG to TAG) is responsible for the defect in these cattle, the only known animal model of the disease (B. Zhang, P. J. Healy, Y. Zhao, D. W. Crabb and R. A. Harris, J. Biol. chem., in press (1990)). This mutation introduces a stop codon in the leader peptide of the EI~ subunit, resulting in premature termination of translation and apparently destabilization of the mRNA. Some patients with maple syrup urine disease will respond to thiamine administration with a reduction in blood branched-chain ~-ketoaeids (60). Thiamine administration may increase tissue levels of thiamine pyrophosphate which may compensate for defective binding of this