Kinetics and specificity of reductive acylation of lipoyl domains from 2-oxo acid dehydrogenase multienzyme complexes.
Lipoamide and a peptide, Thr-Val-Glu-Gly-Asp-Lys-Ala-Ser-Met-Glu lipoylated on the N6-amino group of the lysine residue, were tested as substrates for reductive acetylation by the pyruvate decarboxylase (E1p) component of the pyruvate dehydrogenase multienzyme complex of Escherichia coli. The peptide has the same amino acid sequence as that surrounding the three lipoyllysine residues in the lipoate acetyltransferase (E2p) component of the native enzyme complex. Lipoamide was shown to be a very poor substrate, with a Km much higher than 4 mM and a value of kcat/Km of 1.5 M-1.s-1. Under similar conditions, the three E2p lipoyl domains, excised from the pyruvate dehydrogenase complex by treatment with Staphylococcus aureus V8 proteinase, could be reductively acetylated by E1p much more readily, with a typical Km of approximately 26 microM and a typical kcat of approximately 0.8 s-1. The value of kcat/Km for the lipoyl domains, approximately 3.0 x 10(4) M-1.s-1, is about 20,000 times higher than that for lipoamide as a substrate. This indicates the great improvement in the effectiveness of lipoic acid as a substrate for E1p that accompanies the attachment of the lipoyl group to a protein domain. The free E2o lipoyl domain was similarly found to be capable of being reductively succinylated by the 2-oxoglutarate decarboxylase (E1o) component of the 2-oxoglutarate dehydrogenase complex of E. coli. The 2-oxo acid dehydrogenase complexes are specific for their particular 2-oxo acid substrates. The specificity of the E1 components was found to extend also to the lipoyl domains.(ABSTRACT TRUNCATED AT 250 WORDS)
Graham
L D
LD
Department of Biochemistry, University of Cambridge, England.
Packman
L C
LC
Perham
R N
RN
D042942:3-Methyl-2-Oxobutanoate Dehydrogenase (Lipoamide); D000215:Acylation; D004926:Escherichia coli / Q000201:enzymology; D007658:Ketone Oxidoreductases / Q000378:metabolism*; D007700:Kinetics; D009097:Multienzyme Complexes / Q000378:metabolism*; D010084:Oxidation-Reduction; D013379:Substrate Specificity
D009097:Multienzyme Complexes; D007658:Ketone Oxidoreductases; D042942:3-Methyl-2-Oxobutanoate Dehydrogenase (Lipoamide)