Aspartate-27 and glutamate-473 are involved in catalysis by Zymomonas mobilis pyruvate decarboxylase

Zymomonas mobilis pyruvate decarboxylase (EC 4.1.1.1) was subjected to site-directed mutagenesis at two acidic residues near the thiamin diphosphate cofactor in the active site. Asp-27 was changed to Glu or Asn, and Glu-473 was mutated to Asp (E473D) or Gin (E473Q). Each mutant protein was purified to near-homogeneity, and the kinetic and cofactor-binding properties were compared with those of the wild-type protein. Despite the very conservative nature of these alterations, all mutants had a very low, but measurable, specific activity ranging from 0.025 % (E473Q) to 0.173 % (E473D) of the wild type. With the exception of E473Q, the mutants showed small decreases in the affinity for thiamin diphosphate, and binding of the second cofactor (Mg2+) was also weakened somewhat. With E473Q, both cofactors seemed to be very tightly bound so that they were not removed by the treatment that was effective for the wild-type enzyme and other mutant forms. All mutants showed minor changes in the K-m for substrate, but these alterations did not account for the low activities. These low specific activities, accompanied by little change in the K-m for pyruvate, are consistent with a quantitative model of the catalytic cycle in which the main effect of the mutations is to slow the decarboxylation step with a minor change in the rate constant for pyruvate binding.