Effects of substitution of aspartate‐440 and tryptophan‐487 in the thiamin diphosphate binding region of pyruvate decarboxylase from Zymomonas mobilis

Publisher Website
Google Scholar
Abstract
A tryptophan residue at position 487 in Zymomonas mobilis pyruvate decarboxylase was altered to leucine by site‐directed mutagenesis. This modified Z. mobilis pyruvate decarboxylase was active when expressed in Escherichia coli and had unchanged kinetics towards pyruvate. The enzyme showed a decreased affinity for the cofactors with the half‐saturating concentrations increasing from 0.64 to 9.0 μM for thiamin diphosphate and from 4.21 to 45 μM for Mg2+. Unlike the wild‐type enzyme, there was little quenching of tryptophan fluorescence upon adding, cofactors to this modified form. The data suggest that tryptophan‐487 is close to the cofactor binding site but is not required absolutely for pyruvate decarboxylase activity. Substitution of asparagine, threonine of glycine for aspartate‐440, a residue which is conserved between many thiamin diphosphate‐dependent enzymes, completely abolishes enzyme activity.