| Abstract
| - Pyruvate decarboxylase from yeast (YPDC, EC 4.1.1.1) exhibits a marked lag phase in theprogress curves of product (acetaldehyde) formation. The currently accepted kinetic model for YPDCpredicts that, only upon binding of substrate in a regulatory site, a slow activation step converts inactiveenzyme into the active form. This allosteric behavior gives rise to sigmoidal steady-state kinetics. TheE477Q active site variant of YPDC exhibited hyperbolic initial rate curves at low pH, not consistent withthe model. Progress curves of product formation by this variant were S-shaped, consistent with the presenceof three interconverting conformations with distinct steady-state rates. Surprisingly, wild-type YPDC atpH ≤5.0 also possessed S-shaped progress curves, with the conformation corresponding to the middlesteady state being the most active one. Reexamination of the activation by substrate of wild-type YPDCin 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 curveappears “normal” or S-shaped with an inflection point. The substrate dependence of the apparent rateconstants suggested that the first transition corresponded to substrate binding in an active site and asubsequent step responsible for conversion to an asymmetric conformation. Consequently, the secondenzyme state may report on “unregulated” enzyme, since the regulatory site does not participate in itsgeneration. This enzyme state utilizes the alternating sites mechanism, resulting in the hyperbolic substratedependence of initial rate. The second transition corresponds to binding a substrate molecule in theregulatory site and subsequent minor conformational adjustments. The third enzyme state corresponds tothe allosterically regulated conformation, previously referred to as activated enzyme. The pH dependenceof the Hill coefficient suggests a random binding of pyruvate in a regulatory and an active site of wild-type YPDC. Addition of pyruvamide or acetaldehyde to YPDC results in the appearance of additionalconformations of the enzyme.
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