Pyruvate decarboxylase from Zymomonas mobilis. Structure and re-activation of apoenzyme by the cofactors thiamin diphosphate and magnesium ion.
To study the mechanism of re-activation of Zymomonas mobilis pyruvate decarboxylase apoenzyme by its cofactors thiamin diphosphate and Mg2+, cofactor-free enzyme was prepared by dialysis against 1 mM-dipicolinic acid at pH 8.2. This apoenzyme was then used in a series of experiments that included determination of: (a) the affinity towards one cofactor when the other was present at saturating concentrations; (b) cofactor-binding rates by measuring the quenching of tryptophan fluorescence on the apoenzyme; (c) the effect of replacement of cofactors with various analogues; (d) the stoichiometry of bound cofactors in holoenzyme; and (e) the molecular mass of apoenzyme by gel filtration. The results of these experiments form the basis for a proposed model for the re-activation of Z. mobilis pyruvate decarboxylase apoenzyme by its cofactors. In this model there exists two alterative but equivalent pathways for cofactor binding. In each pathway the first step is an independent reversible binding of either thiamin diphosphate (Kd 187 microM) or Mg2+ (Kd 1.31 mM) to free apoenzyme. When both cofactors are present, the second cofactor-binding step to form active holoenzyme is a slow quasi-irreversible step. This second binding step is a co-operative process for both thiamin diphosphate (Kd 0.353 microM) and Mg2+ (Kd 2.47 microM). Both the apo- and the holo-enzyme have a tetrameric subunit structure, with cofactors binding in a 1:1 ratio with each subunit.
Diefenbach
R J
RJ
Department of Biochemistry, University of Queensland, Australia.
Duggleby
R G
RG
D001051:Apoenzymes / Q000378:metabolism*; D001421:Bacteria, Anaerobic / Q000201:enzymology; D004789:Enzyme Activation; D005033:Ethylmaleimide / Q000494:pharmacology; D006090:Gram-Negative Bacteria / Q000201:enzymology*; D007700:Kinetics; D008274:Magnesium / Q000494:pharmacology*; D008433:Mathematics; D008954:Models, Biological; D011767:Pyruvate Decarboxylase / Q000302:isolation & purification / Q000378:metabolism*; D013835:Thiamine Pyrophosphate / Q000494:pharmacology*
D001051:Apoenzymes; D011767:Pyruvate Decarboxylase; D008274:Magnesium; D005033:Ethylmaleimide; D013835:Thiamine Pyrophosphate