Kinetic characterization of branched chain ketoacid dehydrogenase.
Initial velocity and product inhibition experiments were performed to characterize the kinetic mechanism of branched chain ketoacid dehydrogenase (the branched chain complex) activity. The results were directly compared to predicted patterns for a three-site ping-pong mechanism. Product inhibition experiments confirmed that NADH is competitive versus NAD+ and isovaleryl CoA is competitive versus CoA. Furthermore, both NADH and isovaleryl CoA were uncompetitive versus ketoisovaleric acid. These results are consistent with a ping-pong mechanism and are similar to pyruvate dehydrogenase and alpha-ketoglutarate dehydrogenase. However, inhibition patterns for isovaleryl CoA versus NAD+ and NADH versus CoA are not consistent with a ping-pong mechanism. These patterns may result from a steric interaction between the flavoprotein and transacetylase subunits of the complex. To determine the kinetic mechanism of the substrates and feedback inhibitors (NADH and isovaleryl CoA) of the branched chain complex, it was necessary to define the interaction of the inhibitors at nonsaturating fixed substrate (CoA and NAD+) concentrations. While the competitive inhibition patterns were maintained, slope replots for NADH versus NAD+ at nonsaturating CoA concentrations were parabolic. This unexpected finding resembles a linear mixed type of inhibition where the inhibition is a combination of pure competitive and noncompetitive inhibition.
Boyer
B
B
Louisiana State University Medical Center, Department of Physiology, New Orleans 70112-1393.
Odessey
R
R
D042942:3-Methyl-2-Oxobutanoate Dehydrogenase (Lipoamide); D000818:Animals; D001667:Binding, Competitive; D005420:Flavoproteins / Q000378:metabolism; D007658:Ketone Oxidoreductases / Q000378:metabolism*; D007700:Kinetics; D008928:Mitochondria / Q000201:enzymology*; D009097:Multienzyme Complexes / Q000378:metabolism*; D009243:NAD / Q000378:metabolism; D051381:Rats; D011919:Rats, Inbred Strains
D005420:Flavoproteins; D009097:Multienzyme Complexes; D009243:NAD; D007658:Ketone Oxidoreductases; D042942:3-Methyl-2-Oxobutanoate Dehydrogenase (Lipoamide)