Kinetic mechanism of pyruvate decarboxylase. Evidence for a specific protonation of the enzymic intermediate.
Decarboxylation of pyruvate by pyruvate decarboxylase (EC 4.1.1.1) was performed in a reaction mixture containing 50% deuterium. The isolated product, acetaldehyde, was investigated directly by 1H NMR and by mass spectrometry after conversion to the 2,4-dinitrophenyl hydrazone. The protium content of 56% at acetaldehyde C1 demonstrates a specific protonation of the corresponding intermediate by the enzyme. Proton inventory studies and enzyme modification indicate the 4' amino group of the coenzyme, thiamine pyrophosphate, in an immonium structure being a possible proton donor. A 'partially concerted' mechanism is suggested for the reaction steps following the decarboxylation.
Ermer
J
J
Martin-Luther-University Halle-Wittenberg, Institute of Biochemistry, Germany.
Schellenberger
A
A
Hübner
G
G
D000079:Acetaldehyde / Q000737:chemistry; D007700:Kinetics; D009682:Magnetic Resonance Spectroscopy; D013058:Mass Spectrometry; D011522:Protons; D011767:Pyruvate Decarboxylase / Q000737:chemistry*; D012441:Saccharomyces cerevisiae / Q000201:enzymology
D011522:Protons; D011767:Pyruvate Decarboxylase; D000079:Acetaldehyde