Consequences of a modified putative substrate-activation site on catalysisby yeast pyruvate decarboxylase

Earlier, it had been proposed in the laboratories at Halle that a cysteine residue is responsible for the hysteretic substrate activation behavior of yeast pyruvate decarboxylase. More recently, this idea has received support in a series of studies from Rutgers with the identification of residue C22 1 as the site where substrate is bound to transmit the information to H92, to E91, to W412, and finally to the active center thiamin diphosphate. Acco rding to steady-state kinetic assays, the C221A/C222A variant is no longer subject to substrate activation yet is still a well-functioning enzyme. Sev eral further experiments are reported on this variant: (I) The variant exhi bits lag phases in the product formation progress curves, which can be attr ibuted to a unimolecular step in the pre-steady-state stage of catalysis. ( 2) The rate of exchange with solvent deuterium of the thiamin diphosphate C 2H atom is slowed by a factor of 2 compared to the wild-type enzyme, sugges ting that the reduced activity that results from the substitutions some 20 Angstrom from the active center is also seen in the first key step of the r eaction. (3) The solvent (deuterium oxide) kinetic isotope effect was found to be inverse on V-max/K-m (0.62), and small but normal on V-max (1.26), v irtually ruling out residue C221 as being responsible for the inverse effec ts reported for the wildtype enzyme at low substrate concentrations. The so lvent kinetic isotope effects are compared to those on two related enzymes not subject to substrate activation, Zymomonas mobilis pyruvate decarboxyla se and benzoylformate decarboxylase.