Conversion of Escherichia coli pyruvate oxidase to an 'alpha-ketobutyrate oxidase'

Escherichia coli pyruvate oxidase (PoxB), a lipid-activated homotetrameric enzyme, is active on both pyruvate and 2-oxobutanoate ('alpha -ketobutyrate '), although pyruvate is the favoured substrate. By localized random mutage nesis of residues chosen on the basis of a modelled active site, we obtaine d several PoxB enzymes that had a markedly decreased activity with the natu ral substrate, pyruvate, but retained full activity with 2-oxobutanoate. In each of these mutant proteins VaI-380 had been replaced with a smaller res idue, namely alanine, glycine or serine. One of these, PoxB V380A/L253F, wa s shown to lack detectable pyruvate oxidase activity in vivo; this protein was purified, studied and found to have a 6-fold increase in K-m for pyruva te and a 10-fold lower V-max with this substrate. In contrast, the mutant h ad essentially normal kinetic constants with 2-oxobutanoate. The altered su bstrate specificity was reflected in a decreased rate of pyruvate binding t o the latent conformer of the mutant protein owing to the V380A mutation. T he L253F mutation alone had no effect on PoxB activity, although it increas ed the activity of proteins carrying substitutions at residue 380, as it di d that of the wild-type protein. The properties of the V380A/L253F protein provide new insights into the mode of substrate binding and the unusual act ivation properties of this enzyme.