Role of Glu51 for cofactor binding and catalytic activity in pyruvate decarboxylase from yeast studied by site-directed mutagenesis.
We investigated the importance of the interaction between the Nl'-atom of the cofactor thiamine diphosphate and glutamic acid residue 51 in pyruvate decarboxylase (EC 4.1. 1.1). The yeast wild type gene PDCl and the respective mutant genes (E51Q and E51A) were expressed in Escherichia coli. The three enzymes were purified to homogeneity. They comigrated as a single band during silver-stained SDS/PAGE with a molecular mass of 60 000 Da. A molecular mass of 61 200 +/- 200 Da was determined by mass spectrometry for the subunit. The native enzyme is a homotetramer as demonstrated by gel filtration experiments. Near- and far-UV CD spectra showed no significant differences for the apoenzyme of the wild type and the mutants. Slight differences in the rate of thiamine diphosphate binding to the apoprotein component were observed between the wild type and the E51Q PDC by CD spectroscopy. Compared to the wild type enzyme, thiamine diphosphate binding at the E51A mutant apoprotein is very slow. Only 0.04% of the catalytic activity of the wild type enzyme was observed for the E51Q mutant; the E51A mutant has no detectable catalytic activity. The S0.5 value for the substrate pyruvate is increased 33-fold for the E51Q mutant. Substrate activation was observed for both the wild type and the E51Q mutant. The interaction between the N1'-atom of the coenzyme and glutamic acid 51 strongly influences the catalytic activity but only moderately the binding of the cofactor to the apoenzyme and the substrate activation rate.
Killenberg-Jabs
M
M
Institut für Biochemie, Martin-Luther-Universität Halle-Wittenberg, Halle/Saale, Germany.
König
S
S
Eberhardt
I
I
Hohmann
S
S
Hübner
G
G
D001665:Binding Sites / Q000235:genetics; D002384:Catalysis; D004926:Escherichia coli / Q000235:genetics; D005798:Genes, Bacterial; D005822:Genetic Vectors; D005973:Glutamine / Q000235:genetics* / Q000502:physiology*; D016297:Mutagenesis, Site-Directed; D011767:Pyruvate Decarboxylase / Q000235:genetics* / Q000302:isolation & purification / Q000378:metabolism*; D012441:Saccharomyces cerevisiae / Q000201:enzymology* / Q000235:genetics
D005973:Glutamine; D011767:Pyruvate Decarboxylase