Kinetic plots of acyl-CoA rearrangement activities catalyzed by reconstituted HcmA and HcmB.
A and B, conversion of 2-hydroxyisobutyryl- and (S)-3-hydroxybutyryl-CoA (A) and (R)-3-hydroxybutyryl-, butyryl-, and isobutyryl-CoA by wild-type HcmAB (B). C, conversion of 2-hydroxyisobutyryl- and (S)-3-hydroxybutyryl-CoA by HcmA I90V mutant reconstituted with the wild-type HcmB. All enzyme activities were measured at pH 6.6 and 30 °C. By using nonlinear regression analysis (Graph Pad Prism 5.0 software), Km and Vmax values were revealed (Table 1), applying either the Michaelis-Menten equation (A and B) or an allosteric sigmoidal model (C).