Kinetics and specificity of reductive acylation of wild-type and mutated lipoyl domains of 2-oxo-acid dehydrogenase complexes from Azotobacter vinelandii

Eur J Biochem. 1998 Feb 15;252(1):45-50. doi: 10.1046/j.1432-1327.1998.2520045.x.


The kinetics and specificity of reductive acylation of lipoyl domains derived from Azotobacter vinelandii 2-oxo-acid dehydrogenase complexes, catalysed by A. vinelandii and Escherichia coli complexes, have been investigated. With the wild-type pyruvate dehydrogenase complex from A. vinelandii the rate of reductive acetylation and deacetylation was studied by rapid mixing methods. The rate of reductive acetylation, 126 s(-1), corresponds well with the turnover rate derived from steady-state measurements. Deacetylation was rapid and specific for coenzyme A. No deacetylation was observed with reduced or oxidised lipoamide or with dithiothreitol. The rate of reductive acetylation of complex-bound lipoyl domains by pyruvate dehydrogenase (E1p) is at least 60 times higher than of free lipoyl domains under comparable conditions. This gain in catalytic rate indicates a large diffusion limitation of lipoyl domains when attached via the flexible linker segments to the complex, and illustrates the efficiency of substrate channeling in the multienzyme complex. The 2-oxo-acid dehydrogenases exhibit specificity for lipoyl domains in the reductive acylation reaction. The A. vinelandii lipoyl domain derived from the pyruvate dehydrogenase complex is a good substrate for A. vinelandii E1p, but not for A. vinelandii 2-oxoglutarate dehydrogenase (E1o), and vice versa. The A. vinelandii lipoyl domain of the pyruvate dehydrogenase complex is also, although at a lower rate, reductively acetylated by E. coli E1p and reductively succinylated by E. coli E1o. Likewise, the A. vinelandii lipoyl domain derived from the 2-oxoglutarate dehydrogenase complex is recognised by E. coli E1o, but not by E. coli E1p. This suggests that common determinants of the lipoyl domains exist that are responsible for recognition by the E1 components. On the basis of the observed specificity and lipoyl domain sequences and structures, an exposed loop of the A. vinelandii 2-oxoglutarate dehydrogenase complex lipoyl domain was subjected to mutagenesis. Although the reductive acylation experiments of mutants of the lipoyl domain indicate the importance of this loop for recognition, it is probably not the single determinant for specificity.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • 3-Methyl-2-Oxobutanoate Dehydrogenase (Lipoamide)
  • Acetates / metabolism
  • Acylation
  • Amino Acid Sequence
  • Azotobacter vinelandii / enzymology*
  • Bacterial Proteins / metabolism
  • Bacterial Proteins / physiology
  • Binding Sites
  • Escherichia coli / enzymology
  • Ketoglutarate Dehydrogenase Complex / chemistry
  • Ketoglutarate Dehydrogenase Complex / metabolism
  • Ketone Oxidoreductases / metabolism*
  • Kinetics
  • Models, Molecular
  • Molecular Sequence Data
  • Multienzyme Complexes / metabolism*
  • Mutagenesis, Site-Directed / genetics
  • Oxidation-Reduction
  • Pyruvate Dehydrogenase Complex / metabolism
  • Sequence Alignment
  • Substrate Specificity
  • Thioctic Acid / analogs & derivatives
  • Thioctic Acid / metabolism


  • Acetates
  • Bacterial Proteins
  • Multienzyme Complexes
  • Pyruvate Dehydrogenase Complex
  • Thioctic Acid
  • Ketone Oxidoreductases
  • Ketoglutarate Dehydrogenase Complex
  • 3-Methyl-2-Oxobutanoate Dehydrogenase (Lipoamide)