Roles of amino acid residues surrounding phosphorylation site 1 of branched-chain α-ketoacid dehydrogenase (BCKDH) in catalysis and phosphorylation site recognition by BCKDH kinase

John W. Hawes, R. Jason Schnepf, Anne E. Jenkins, Yoshiharu Shimomura, Kirill M. Popov, Robert A. Harris

Research output: Contribution to journalArticle

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Abstract

Branched-chain α-ketoacid dehydrogenase is regulated by reversible phosphorylation of serine 293 (site 1) on the E1α subunit. Alanine-scanning mutagenesis was used to examine the roles of residues surrounding serine 293 in catalysis by the dehydrogenase and in substrate recognition by branched- chain α-ketoacid dehydrogenase kinase. Alanine substitution of serine 293 resulted in a 10-fold increased K(m) for α-ketoisovalerate, a less increased (2.8-fold) K(m) for α-ketoisocaproate, but no change in V(max) or the K(m) for thiamine pyrophosphate. Alanine substitutions of arginine 288, histidine 292, and aspartate 296, residues highly conserved among α-ketoacid dehydrogenases, resulted in inactive enzymes. Each of the inactive E1 mutants bound to the E2 core subunit with equal affinity as wild-type E1, and each produced circular dichroism spectra identical to that of wild-type E1. Two mutations, H292A and S293E, abolished the ability of E1 apoenzyme to reconstitute with thiamine pyrophosphate. Each alanine-substituted E1 was phosphorylated at site 1 by branched-chain α-ketoacid dehydrogenase kinase with similar rates, with the exception of the R288A mutant, which displayed no detectable phosphorylation. Thiamine pyrophosphate inhibited the phosphorylation of all mutant enzymes with the exception of H292A, the mutant E1 that did not bind thiomine pyrophosphate.

Original languageEnglish (US)
Pages (from-to)31071-31076
Number of pages6
JournalJournal of Biological Chemistry
Volume270
Issue number52
DOIs
StatePublished - Dec 29 1995

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3-Methyl-2-Oxobutanoate Dehydrogenase (Lipoamide)
Phosphorylation
Thiamine Pyrophosphate
Catalysis
Alanine
Phosphotransferases
Serine
Amino Acids
Oxidoreductases
Substitution reactions
Apoenzymes
Mutagenesis
Enzymes
Circular Dichroism
Histidine
Aspartic Acid
Arginine
Scanning
Mutation
Substrates

ASJC Scopus subject areas

  • Biochemistry
  • Molecular Biology
  • Cell Biology

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Roles of amino acid residues surrounding phosphorylation site 1 of branched-chain α-ketoacid dehydrogenase (BCKDH) in catalysis and phosphorylation site recognition by BCKDH kinase. / Hawes, John W.; Schnepf, R. Jason; Jenkins, Anne E.; Shimomura, Yoshiharu; Popov, Kirill M.; Harris, Robert A.

In: Journal of Biological Chemistry, Vol. 270, No. 52, 29.12.1995, p. 31071-31076.

Research output: Contribution to journalArticle

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abstract = "Branched-chain α-ketoacid dehydrogenase is regulated by reversible phosphorylation of serine 293 (site 1) on the E1α subunit. Alanine-scanning mutagenesis was used to examine the roles of residues surrounding serine 293 in catalysis by the dehydrogenase and in substrate recognition by branched- chain α-ketoacid dehydrogenase kinase. Alanine substitution of serine 293 resulted in a 10-fold increased K(m) for α-ketoisovalerate, a less increased (2.8-fold) K(m) for α-ketoisocaproate, but no change in V(max) or the K(m) for thiamine pyrophosphate. Alanine substitutions of arginine 288, histidine 292, and aspartate 296, residues highly conserved among α-ketoacid dehydrogenases, resulted in inactive enzymes. Each of the inactive E1 mutants bound to the E2 core subunit with equal affinity as wild-type E1, and each produced circular dichroism spectra identical to that of wild-type E1. Two mutations, H292A and S293E, abolished the ability of E1 apoenzyme to reconstitute with thiamine pyrophosphate. Each alanine-substituted E1 was phosphorylated at site 1 by branched-chain α-ketoacid dehydrogenase kinase with similar rates, with the exception of the R288A mutant, which displayed no detectable phosphorylation. Thiamine pyrophosphate inhibited the phosphorylation of all mutant enzymes with the exception of H292A, the mutant E1 that did not bind thiomine pyrophosphate.",
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