Gary W. Goodwin

Regulation of branched-chain α-ketoacid dehydrogenase complex by covalent modification

Abstract The branched-chain α-ketoacid dehydrogenase complex, like the pyruvate dehydrogenase complex, is an intramitochondrial enzyme subject to regulation by covalent modification. Phosphorylation causes inactivation and dephosphorylation causes activation of both complexes. The branched-chain α-ketoacid dehydrogenase kinase, believed distinct from pyruvate dehydrogenase kinase, is an integral component of the branched-chain α-ketoacid dehydrogenase complex and is sensitive to inhibition by branched-chain α-ketoacids, α-chloroisocaproate, phenylpyruvate, clofibric acid, octanoate and dichloroacetate. Phosphorylation of branched-chain α-ketoacid dehydrogenase occurs at two closely-linked serine residues (sites 1 and 2) of the α-subunit of the decarboxylase. HPLC and sequence data suggest homology of the amino acid sequence adjacent to phosphorylation sites 1 and 2 of complexes isolated from several different tissues. Stoichiometry for phosphorylation of all of the complexes studies was about 1 mol P/mol α-subunit for 95% inactivation and 1.5 mol P/mol α-subunit for maximally phosphorylated complex. Site 1 and site 2 were phosphorylated at similar rates until total phosphorylation exceeded 1 mol P/mol α-subunit. The complexes from rabbit kidney, rabbit heart, and rat heart showed 30–40% additional phosphorylation of the α-subunit beyond 95% inactivation. Site specificity studies carried out with the kinase partially inhibited with α-chloroisocaproate suggest that phosphorylation of site 1 is primarily responsible for regulation of the complex. The capacity of the branched-chain α-ketoacid dehydrogenase to oxidize pyruvate ( K m = 0.8 m m , V max = 20% of that of α-ketoisovalerate) interferes with the estimation of activity state of the hepatic pyruvate dehydrogenase complex. The disparity between the activity states of the two complexes in most physiologic states contributes to this interference. An inhibitory antibody for branched-chain α-ketoacid dehydrogenase can be used to prevent interference with the pyruvate dehydrogenase assay. Almost all of the hepatic branched-chain α-ketoacid dehydrogenase in chow-fed rats is active (> 90% dephosphorylated). In contrast, almost all of the hepatic enzyme of rats fed a low-protein (8%) diet is inactive (> 85% phosphorylated). Fasting of chow-fed rats has no effect on the activity state of hepatic branched-chain α-ketoacid dehydrogenase, i.e. > 90% of the enzyme remains in the active state. However, fasting of rats maintained on low-protein diets greatly activates the hepatic enzyme. Thus, dietary protein deficiency results in inactivation of hepatic branched-chain α-ketoacid dehydrogenase, presumably because of low hepatic levels of branched-chain α-ketoacids, established inhibitors of branched-chain α-ketoacid dehydrogenase kinase. With rats fed a low-protein diet and subsequently fasted, inhibition of branched-chain α-ketoacid dehydrogenase kinase by branched-chain α-ketoacids generated from branched-chain amino acid produced by proteolysis of endogenous protein most likely accounts for the greater activity state of the branched-chain α-ketoacid dehydrogenase complex. Hepatocytes isolated from rats fed a chow diet or a low-protein (8%) diet were used to study the activity state and flux through the branched-chain α-ketoacid dehydrogenase complex. Alpha-chloroisocaproate stimulated α-ketoisovalerate decarboxylation with hepatocytes from rats fed a low-protein diet but had no effect with hepatocytes from rats fed chow diet. Activity measurements indicated that branched-chain α-ketoacid dehydrogenase was mainly in the inactive (phosphorylated) state in hepatocytes from low-protein-fed rats but mainly in the active (dephosphorylated) state in hepatocytes from chow-fed rats. Furthermore, α-ketoisocaproate greatly activated ( A 50 = 20 μ m ) α-ketoisovalerate oxidation by hepatocytes isolated from low-protein-fed rats but had no effect with hepatocytes isolated from chow-fed rats. The dietary studies and the hepatocyte experiments, taken together, suggest that portal blood levels of branched-chain α-ketoacids, particularly α-ketoisocaproate,are important determinants of the activity state of the hepatic branched-chain α-ketoacid dehydrogenase complex.

Regulation of the branched-chain α-ketoacid dehydrogenase and elucidation of a molecular basis for maple syrup urine disease

The hepatic branched-chain alpha-ketoacid dehydrogenase complex plays an important role in regulating branched-chain amino acid levels. These compounds are essential for protein synthesis but toxic if present in excess. When dietary protein is deficient, the hepatic enzyme is converted to the inactive, phosphorylated state to conserve branched-chain amino acids for protein synthesis. When dietary protein is excessive, the enzyme is in the active, dephosphorylated state to commit the excess branched-chain amino acids to degradation. Inhibition of protein synthesis by cycloheximide, even when the animal is starving for dietary protein, results in activation of the hepatic branched-chain alpha-ketoacid dehydrogenase complex to prevent accumulation of branched-chain amino acids. Likewise, the increase in branched-chain amino acids caused by body wasting during starvation and uncontrolled diabetes is blunted by activation of the hepatic branched-chain alpha-ketoacid dehydrogenase complex. The activity state of the complex is regulated in the short term by the concentration of branched-chain alpha-ketoacids (inhibitors of branched-chain alpha-ketoacid dehydrogenase kinase) and in the long term by alteration in total branched-chain alpha-ketoacid dehydrogenase kinase activity. cDNAs have been cloned and the primary structure of the mature proteins deduced for the E1 alpha subunit of the human and rat liver branched-chain alpha-ketoacid dehydrogenase complex. The cDNA and protein sequences are highly conserved for the two species. Considerable sequence similarity is also apparent between the E1 alpha subunits of the human branched-chain alpha-ketoacid dehydrogenase complex and the pyruvate dehydrogenase complex. Maple syrup urine disease is caused by an inherited deficiency in the branched-chain alpha-ketoacid dehydrogenase complex. The molecular basis of one maple syrup urine disease family has been determined for the first time. The patient was found to be a compound heterozygote, inheriting an allele encoding an abnormal E1 alpha from the father, and an allele which is not expressed from the mother. The only known animal model for the disease (Polled Hereford cattle) has also been characterized. The mutation in these animals introduces a stop codon in the leader peptide of the E1 alpha subunit, resulting in premature termination of translation. Two thiamine responsive patients have been studied. The deduced amino acid sequences of the mature E1 alpha subunit and its leader sequence were normal, suggesting that the defect in these patients must exist in some other subunit of the complex. 3-Hydroxyisobutyrate dehydrogenase and methylmalonate-semialdehyde dehydrogenase, two enzymes of the valine catabolic pathway, were purified from liver tissue and characterized.(ABSTRACT TRUNCATED AT 400 WORDS)

Enzymatic determination of the branched-chain α-keto acids

A spectrophotometric endpoint assay for determination of branched-chain alpha-keto acids is described. The assay depends on measurement of the NADH produced after addition of branched-chain alpha-keto acid dehydrogenase. Interference by pyruvate and alpha-ketobutyrate was eliminated by pretreating the sample with pyruvate dehydrogenase. The method yielded a peripheral venous plasma value of 59 +/- 5 microM (mean +/- SE) for the branched-chain alpha-keto acids of five overnight fasted healthy humans.

Nutritional and Hormonal Regulation of the Activity State of Hepatic Branched-Chain α-Keto Acid Dehydrogenase Complexa

The hepatic branched-chain alpha-keto acid dehydrogenase complex plays an important role in regulating branched-chain amino acid levels. These compounds are essential for protein synthesis but are toxic if present in excess. When dietary protein is deficient, the hepatic enzyme is present in the inactive, phosphorylated state to allow conservation of branched-chain amino acids for protein synthesis. When dietary protein is excessive, the enzyme is in the active, dephosphorylated state to commit the excess branched-chain amino acids to degradation. Inhibition of protein synthesis by cycloheximide, even when the animal is starving for protein, results in activation of the hepatic branched-chain alpha-keto acid dehydrogenase complex to prevent accumulation of branched-chain amino acids. Likewise, the increase in branched-chain amino acids caused by body wasting during starvation and uncontrolled diabetes is blunted by activation of the hepatic branched-chain alpha-keto acid dehydrogenase complex. The activity state of the hepatic branched-chain alpha-keto acid dehydrogenase complex is regulated in the short term by the concentration of branched-chain alpha-keto acids (inhibitors of branched-chain alpha-keto acid dehydrogenase kinase) and in the long term by alteration in the total branched chain alpha-keto acid dehydrogenase kinase activity.

Activation of branched-chain α-ketoacid dehydrogenase in isolated hepatocytes by branched-chain α-ketoacids

Abstract The effects of branched-chain α-ketoacids on flux through and activity state of the branched-chain α-ketoacid dehydrogenase complex were studied in hepatocytes prepared from chow-fed, starved, and low-protein-diet-fed rats. Very low concentrations of α-ketoisocaproate caused a dramatic stimulation (50% activation at 20 μ m ) of α-ketoisovalerate decarboxylation in hepatocytes from low-protein-fed rats. α-Keto-β-methylvalerate was also effective, but less so than α-ketoisocaproate. α-Ketoisocaproate did not stimulate α-ketoisovalerate decarboxylation by hepatocytes from chow-fed or starved rats. To a smaller degree, α-keto-β-methylvalerate and α-ketoisovalerate stimulated α-ketoisocaproate decarboxylation by hepatocytes from low-protein-fed rats. The implied order of potency of stimulation of flux through branched-chain α-ketoacid dehydrogenase was α-ketoisocaproate > α-keto-β-methylvalerate > α-ketosiovalerate, i.e., the same order of potency of these compounds as branched-chain α-ketoacid dehydrogenase kinase inhibitors. Fluoride, known to inhibit branched-chain α-ketoacid dehydrogenase phosphatase, largely prevented a-ketoisocaproate and α-chloroisocaproate activation of flux through the branched-chain a-ketoacid dehydrogenase. Assay of the branched-chain α-ketoacid complex in cell-free extracts of hepatocytes isolated from low-protein-diet-fed rats confirmed that α-ketoacids affected the activity state of the complex. Branched-chain α-ketoacids failed to activate flux in hepatocytes prepared from chow-fed and starved rats because essentially all of the complex was already in the dephosphorylated, active state. These findings indicate that inhibition of branched-chain α-ketoacid dehydrogenase kinase activity by branched-chain α-ketoacids is important for regulation of the activity state of hepatic branched-chain α-ketoacid dehydrogenase.

Mammalian methylmalonate-semialdehyde dehydrogenase.

Methylmalonate-semialdehyde dehydrogenase (MMSDH), located in the mitochondrial matrix space, catalyzes the irreversible oxidative decarboxylation of malonate semialdehyde and methylmalonate semialdehyde to acetyl-CoA and propionyl-CoA, respectively. These reactions are in the distal portions of the valine and pyrimidine catabolic pathways. 1,2 MMSDH belongs to the aldehyde dehydrogenase superfamily,3,4 but is unique among the members of this family because coenzyme A is required for the reaction and a CoA ester is produced.1,2 MMSDH, like other aldehyde dehydrogenases, has esterase activity.5 An active site S-acyl enzyme is common to both the esterase reaction and the aldehyde dehydrogenase/CoA ester synthetic reaction.5 An active site cysteine residue (Cys-285 in rat MMSDH3) is acetylated during hydrolysis of p-nitrophenyl acetate by MMSDH. Acetyl-CoA is produced instead of acetate from p-nitrophenyl acetate when the reaction is conducted in the presence of coenzyme A.5 Long-chain fatty acyl-CoA esters also inactivate MMSDH by acylation of its active site cysteine residue,6 presumably by reversal of the above process. Active site acylation has been proposed as a mechanism for the regulation of MMSDH activity in vivo by long-chain fatty acids.7

Spectrophotometric enzymatic assay for S-3-hydroxyisobutyrate☆

An enzymatic spectrophotometric end-point assay has been developed for determination of S-3-hydroxyisobutyrate in biological fluids. The assay measures NADH production at 340 nm after initiation of the reaction with rabbit liver 3-hydroxyisobutyrate dehydrogenase (EC 1.1.1.31). The assay is not affected by R-3-hydroxyisobutyrate, lactate, malate, 3-hydroxybutyrate, 2-methyl-3-hydroxybutyrate, 3-hydroxyisovalerate, 3-hydroxy-n-valerate, 2-methyl-3-hydroxy-valerate, and 3-hydroxypropionate. The assay does measure 2-ethyl-3-hydroxypropionate, a minor metabolite produced by catabolism of alloisoleucine. Application of the method to measure S-3-hydroxyisobutyrate in plasma obtained from normal, 48-h starved, and mildly and severely diabetic rats gave levels of 28, 42, 112, and 155 microM, respectively.

cDNA Cloning of the E1α Subunit of the Branshed-Chain α-Keto Acid Dehydrogenase and Elucidation of a Molecular Basis for Maple Syrup Urine Diseasea

We have cloned cDNAs encoding human and rat liver BCKDH E1 alpha subunits and deduced the primary structure of the mature protein. The sequences of the cDNA and protein are highly conserved between the two species. Significant sequence similarity has also been found between human BCKDH and PDH E1 alpha subunits. We have studied the molecular basis of MSUD by determining the enzyme activity and levels of BCKDH protein and mRNA, and by enzymatic amplification and sequencing of BCKDH E1 alpha-specific mRNA, from an MSUD patient and his parents. Different mutant alleles were identified in the two parents. The patient was a compound heterozygote, inheriting an allele encoding an abnormal E1 alpha from the father and an allele containing a defect in regulation from the mother. Our results demonstrate that a case of MSUD was caused by structural and regulatory mutations involving the E1 alpha subunit.