THE LINKAGE OF CATALYSIS AND REGULATION IN ENZYME ACTION, SOLVENT ISOTOPE EFFECTS AS PROBES OF PROTONIC SITES IN THE YEAST PYRUVATE DECARBOXYLASE MECHANISM

Yeast pyruvate decarboxylase, a thiamin-diphosphate-dependent enzyme w hich undergoes slow hysteretic activation by its own substrate pyruvat e to form an active enzyme that cycles several thousand times before d eactivation, exhibits rate constants (a) 2-fold larger in deuterium ox ide than in protium oxide for the second-order kinetic term in pyruvat e (k/A), (b) 2.3-fold larger in deuterium oxide for the first-order te rm in pyruvate (k/B), and (c) 1.5-fold larger in protium oxide than in deuterium oxide for the zero-order term in pyruvate. Proton inventori es (rates in mixtures of protium and deuterium oxides) for k/A and k/B suggest that the isotope effects arise from addition of an enzymic su lfhydryl group to the regulatory pyruvate preceding the transition sta te for combination of pyruvate with the activated enzyme, with the add ition reaction occurring in every catalytic cycle. The proton inventor y for k is consistent with sulfhydryl addition to the regulatory pyruv ate, coupled to a multiproton process in the transition state for rele ase of the product acetaldehyde. A model for regulation is suggested i n which the opening and closing of sequestering structures at the acti ve site are driven by sulfhydryl addition/elimination reactions at the carbonyl group of the regulatory pyruvate molecule.