Escherichia coli pyruvate oxidase (PoxB), a lipid-activated homotetrameric enzyme, is active on both pyruvate and 2-oxobutanoate (‘α-ketobutyrate’), although pyruvate is the favoured substrate. By localized random mutagenesis of residues chosen on the basis of a modelled active site, we obtained several PoxB enzymes that had a markedly decreased activity with the natural substrate, pyruvate, but retained full activity with 2-oxobutanoate. In each of these mutant proteins Val-380had been replaced with a smaller residue, namely alanine, glycine or serine. One of these, PoxB V380A/L253F, was shown to lack detectable pyruvate oxidase activity in vivo; this protein was purified, studied and found to have a 6-fold increase in Km for pyruvate and a 10-fold lower Vmax with this substrate. In contrast, the mutant had essentially normal kinetic constants with 2-oxobutanoate. The altered substrate specificity was reflected in a decreased rate of pyruvate binding to the latent conformer of the mutant protein owing to the V380A mutation. The L253F mutation alone had no effect on PoxB activity, although it increased the activity of proteins carrying substitutions at residue 380, as it did 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 activation properties of this enzyme.
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Research Article| December 08 2000
Conversion of Escherichia coli pyruvate oxidase to an ‘α-ketobutyrate oxidase’
Ying-Ying CHANG ;
John E. CRONAN, JR
John E. CRONAN, JR 1
*Department of Microbiology, University of Illinois, B103 Chemical and Life Sciences Laboratory, 601 South Goodwin Avenue, Urbana, IL 61801, U.S.A.
†Department of Biochemistry, University of Illinois, B103 Chemical and Life Sciences Laboratory, 601 South Goodwin Avenue, Urbana, IL 61801, U.S.A.
1To whom correspondence should be addressed at the Department of Microbiology (e-mail firstname.lastname@example.org).
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Ying-Ying CHANG, John E. CRONAN; Conversion of Escherichia coli pyruvate oxidase to an ‘α-ketobutyrate oxidase’. Biochem J 15 December 2000; 352 (3): 717–724. doi: https://doi.org/10.1042/bj3520717
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