Activation of thiamine diphosphate in pyruvate decarboxylase from Zymomonas mobilis

Replacement of tryptophan 392 located in the active site cavity of pyruvate decarboxylase (PDC; EC 4.1.1.1) from Zymomonas mobilis by methionine or gl utamine yields enzymes with smaller catalytic constants of 8.5 s(-1) and 3. 6 s(-1) at 4 degrees C, compared to that of the wild-type enzyme (17 s(-1)) . The rate constants of the H/D exchange at the C2 of the coenzyme thiamine diphosphate have been determined to be 130 s(-1) for the wild-type enzyme, 56 s(-1) for the methionine and 30 s(-1) for the glutamine mutant, respect ively, A group with a pK(a) of about 5 has been identified to be essential for C2 deprotonation of the enzyme-bound thiamine diphosphate from the pH d ependence of the H/D exchange. (C) 1998 Federation of European Biochemical Societies.