Abstract
Pyruvate decarboxylase from yeast (YPDC, EC 4.1.1.1) exhibits a marked lag phase in the progress curves of product (acetaldehyde) formation. The currently accepted kinetic model for YPDC predicts that, only upon binding of substrate in a regulatory site, a slow activation step converts inactive enzyme into the active form. This allosteric behavior gives rise to sigmoidal steady-state kinetics. The E477Q active site variant of YPDC exhibited hyperbolic initial rate curves at low pH, not consistent with the model. Progress curves of product formation by this variant were S-shaped, consistent with the presence of three interconverting conformations with distinct steady-state rates. Surprisingly, wild-type YPDC at pH < or =5.0 also possessed S-shaped progress curves, with the conformation corresponding to the middle steady state being the most active one. Reexamination of the activation by substrate of wild-type YPDC in the pH range of 4.5-6.5 revealed two characteristic transitions at all pH values. The values of steady-state rates are functions of both pH and substrate concentration, affecting whether the progress curve appears "normal" or S-shaped with an inflection point. The substrate dependence of the apparent rate constants ...Continue Reading
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Citations
Jan 15, 2009·Biochemistry·Sumit ChakrabortyFrank Jordan
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