Site-directed mutagenesis of phosphorylation sites of the branched chain α-ketoacid dehydrogenase complex

Yu Zhao, John Hawes, Kirill M. Popov, Jerzy Jaskiewicz, Yoshiharu Shimomura, David Crabb, Robert Harris

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Abstract

Regulation of the branched chain α-ketoacid dehydrogenase complex, the rate-limiting enzyme of branched chain amino acid catabolism, involves phosphorylation of 2 amino acid residues (site 1, serine 293; site 2, serine 303). To directly assess the roles played by these sites, site-directed mutagenesis was used to convert these serines to glutamates and/or alanines. Functional E1 heterotetramers were expressed in Escherichia coli carrying genes for E1α and E1β under control of separate T7 promoters in a dicistronic vector. Mutation of phosphorylation site 1 serine to glutamate inactivated E1 activity, i.e. mimicked the effect of phosphorylation of site 1. Replacement of the site 1 serine with alanine greatly increased K(m) for the α-ketoacid substrate but had no effect on maximum velocity. The site 1 serine to alanine mutant was phosphorylated at site 2, but phosphorylation had no effect upon enzyme activity. Mutation of site 2 serine to either glutamate or alanine also had no effect upon enzyme activity, but phosphorylation of these proteins at site 1 inhibited enzyme activity. E1 mutated to change both phosphorylation site serines to glutamates was without enzyme activity. The binding affinity of E1 to the E2 core was not affected by mutation of the phosphorylation sites to glutamates, suggesting no gross perturbation of the association of E1 with the E2 core. The results provide direct evidence that a negative charge at phosphorylation site 1 is responsible for kinase-mediated inactivation of E1. Site 2 is silent with respect to regulation of activity by phosphorylation.

Original languageEnglish
Pages (from-to)18583-18587
Number of pages5
JournalJournal of Biological Chemistry
Volume269
Issue number28
StatePublished - Jul 15 1994

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3-Methyl-2-Oxobutanoate Dehydrogenase (Lipoamide)
Mutagenesis
Phosphorylation
Site-Directed Mutagenesis
Serine
Enzyme activity
Glutamates
Alanine
Enzymes
Mutation
Glutamic Acid
Branched Chain Amino Acids
Escherichia coli
Phosphotransferases
Genes
Association reactions

ASJC Scopus subject areas

  • Biochemistry

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Site-directed mutagenesis of phosphorylation sites of the branched chain α-ketoacid dehydrogenase complex. / Zhao, Yu; Hawes, John; Popov, Kirill M.; Jaskiewicz, Jerzy; Shimomura, Yoshiharu; Crabb, David; Harris, Robert.

In: Journal of Biological Chemistry, Vol. 269, No. 28, 15.07.1994, p. 18583-18587.

Research output: Contribution to journalArticle

Zhao, Yu ; Hawes, John ; Popov, Kirill M. ; Jaskiewicz, Jerzy ; Shimomura, Yoshiharu ; Crabb, David ; Harris, Robert. / Site-directed mutagenesis of phosphorylation sites of the branched chain α-ketoacid dehydrogenase complex. In: Journal of Biological Chemistry. 1994 ; Vol. 269, No. 28. pp. 18583-18587.
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abstract = "Regulation of the branched chain α-ketoacid dehydrogenase complex, the rate-limiting enzyme of branched chain amino acid catabolism, involves phosphorylation of 2 amino acid residues (site 1, serine 293; site 2, serine 303). To directly assess the roles played by these sites, site-directed mutagenesis was used to convert these serines to glutamates and/or alanines. Functional E1 heterotetramers were expressed in Escherichia coli carrying genes for E1α and E1β under control of separate T7 promoters in a dicistronic vector. Mutation of phosphorylation site 1 serine to glutamate inactivated E1 activity, i.e. mimicked the effect of phosphorylation of site 1. Replacement of the site 1 serine with alanine greatly increased K(m) for the α-ketoacid substrate but had no effect on maximum velocity. The site 1 serine to alanine mutant was phosphorylated at site 2, but phosphorylation had no effect upon enzyme activity. Mutation of site 2 serine to either glutamate or alanine also had no effect upon enzyme activity, but phosphorylation of these proteins at site 1 inhibited enzyme activity. E1 mutated to change both phosphorylation site serines to glutamates was without enzyme activity. The binding affinity of E1 to the E2 core was not affected by mutation of the phosphorylation sites to glutamates, suggesting no gross perturbation of the association of E1 with the E2 core. The results provide direct evidence that a negative charge at phosphorylation site 1 is responsible for kinase-mediated inactivation of E1. Site 2 is silent with respect to regulation of activity by phosphorylation.",
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N2 - Regulation of the branched chain α-ketoacid dehydrogenase complex, the rate-limiting enzyme of branched chain amino acid catabolism, involves phosphorylation of 2 amino acid residues (site 1, serine 293; site 2, serine 303). To directly assess the roles played by these sites, site-directed mutagenesis was used to convert these serines to glutamates and/or alanines. Functional E1 heterotetramers were expressed in Escherichia coli carrying genes for E1α and E1β under control of separate T7 promoters in a dicistronic vector. Mutation of phosphorylation site 1 serine to glutamate inactivated E1 activity, i.e. mimicked the effect of phosphorylation of site 1. Replacement of the site 1 serine with alanine greatly increased K(m) for the α-ketoacid substrate but had no effect on maximum velocity. The site 1 serine to alanine mutant was phosphorylated at site 2, but phosphorylation had no effect upon enzyme activity. Mutation of site 2 serine to either glutamate or alanine also had no effect upon enzyme activity, but phosphorylation of these proteins at site 1 inhibited enzyme activity. E1 mutated to change both phosphorylation site serines to glutamates was without enzyme activity. The binding affinity of E1 to the E2 core was not affected by mutation of the phosphorylation sites to glutamates, suggesting no gross perturbation of the association of E1 with the E2 core. The results provide direct evidence that a negative charge at phosphorylation site 1 is responsible for kinase-mediated inactivation of E1. Site 2 is silent with respect to regulation of activity by phosphorylation.

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