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 glutamine 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, respectively. A group with a pKa of about 5 has been identified to be essential for C2 deprotonation of the enzyme-bound thiamine diphosphate from the pH dependence of the H/D exchange.
Tittmann
K
K
Institut für Biochemie, Fachbereich Biochemie/Biotechnologie, Martin-Luther-Universität Halle-Wittenberg, Halle, Germany.
Mesch
K
K
Pohl
M
M
Hübner
G
G
D006863:Hydrogen-Ion Concentration; D007700:Kinetics; D009154:Mutation; D011522:Protons; D011767:Pyruvate Decarboxylase / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D013835:Thiamine Pyrophosphate / Q000378:metabolism*; D017355:Zymomonas / Q000201:enzymology*
D011522:Protons; D011767:Pyruvate Decarboxylase; D013835:Thiamine Pyrophosphate