Ak. Chang et al., Aspartate-27 and glutamate-473 are involved in catalysis by Zymomonas mobilis pyruvate decarboxylase, BIOCHEM J, 339, 1999, pp. 255-260
Zymomonas mobilis pyruvate decarboxylase (EC 4.1.1.1) was subjected to site
-directed mutagenesis at two acidic residues near the thiamin diphosphate c
ofactor in the active site. Asp-27 was changed to Glu or Asn, and Glu-473 w
as mutated to Asp (E473D) or Gin (E473Q). Each mutant protein was purified
to near-homogeneity, and the kinetic and cofactor-binding properties were c
ompared with those of the wild-type protein. Despite the very conservative
nature of these alterations, all mutants had a very low, but measurable, sp
ecific 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 t
he affinity for thiamin diphosphate, and binding of the second cofactor (Mg
2+) was also weakened somewhat. With E473Q, both cofactors seemed to be ver
y tightly bound so that they were not removed by the treatment that was eff
ective 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 accou
nt for the low activities. These low specific activities, accompanied by li
ttle change in the K-m for pyruvate, are consistent with a quantitative mod
el of the catalytic cycle in which the main effect of the mutations is to s
low the decarboxylation step with a minor change in the rate constant for p
yruvate binding.