Yeast pyruvate decarboxylase, a thiamin-diphosphate-dependent enzyme which undergoes slow hysteretic activation by its own substrate pyruvate to form an active enzyme that cycles several thousand times before deactivation, exhibits rate constants (a) 2-fold larger in deuterium oxide than in protium oxide for the second-order kinetic term in pyruvate (k/A), (b) 2.3-fold larger in deuterium oxide for the first-order term 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 inventories (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 sulfhydryl group to the regulatory pyruvate preceding the transition state for combination of pyruvate with the activated enzyme, with the addition reaction occurring in every catalytic cycle. The proton inventory for k is consistent with sulfhydryl addition to the regulatory pyruvate, coupled to a multiproton process in the transition state for release of the product acetaldehyde. A model for regulation is suggested in which the opening and closing of sequestering structures at the active site are driven by sulfhydryl addition/elimination reactions at the carbonyl group of the regulatory pyruvate molecule.