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

Robert Harris, Ralph Paxton, Stephen M. Powell, Gary W. Goodwin, Martha J. Kuntz, Amy C. Han

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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 (Km = 0.8 mm, Vmax = 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 (A50 = 20 μm) 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.

Original languageEnglish
Pages (from-to)219-237
Number of pages19
JournalAdvances in Enzyme Regulation
Volume25
Issue numberC
DOIs
StatePublished - 1986

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3-Methyl-2-Oxobutanoate Dehydrogenase (Lipoamide)
Rats
Phosphorylation
Nutrition
Hepatocytes
Protein-Restricted Diet
Liver
Proteins
Phosphotransferases
Pyruvate Dehydrogenase Complex
Enzymes
Pyruvic Acid
Fasting
Clofibric Acid
Proteolysis
Diet
Rabbits
Amino Acid Sequence Homology
Protein Deficiency
Branched Chain Amino Acids

ASJC Scopus subject areas

  • Biochemistry
  • Molecular Biology

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Regulation of branched-chain α-ketoacid dehydrogenase complex by covalent modification. / Harris, Robert; Paxton, Ralph; Powell, Stephen M.; Goodwin, Gary W.; Kuntz, Martha J.; Han, Amy C.

In: Advances in Enzyme Regulation, Vol. 25, No. C, 1986, p. 219-237.

Research output: Contribution to journalArticle

Harris, Robert ; Paxton, Ralph ; Powell, Stephen M. ; Goodwin, Gary W. ; Kuntz, Martha J. ; Han, Amy C. / Regulation of branched-chain α-ketoacid dehydrogenase complex by covalent modification. In: Advances in Enzyme Regulation. 1986 ; Vol. 25, No. C. pp. 219-237.
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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 (Km = 0.8 mm, Vmax = 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 (A50 = 20 μm) 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.",
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N2 - 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 (Km = 0.8 mm, Vmax = 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 (A50 = 20 μm) 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.

AB - 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 (Km = 0.8 mm, Vmax = 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 (A50 = 20 μm) 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.

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