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 Gln (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 Km for substrate, but these alterations did not account for the low activities. These low specific activities, accompanied by little change in the Km 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.
Chang
A K
AK
Centre for Protein Structure, Function and Engineering, Department of Biochemistry, The University of Queensland, Brisbane, Qld. 4072, Australia.
Nixon
P F
PF
Duggleby
R G
RG
D001224:Aspartic Acid / Q000378:metabolism*; D002384:Catalysis; D004591:Electrophoresis, Polyacrylamide Gel; D018698:Glutamic Acid / Q000378:metabolism*; D007700:Kinetics; D016297:Mutagenesis, Site-Directed; D011487:Protein Conformation; D011767:Pyruvate Decarboxylase / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D013379:Substrate Specificity; D013835:Thiamine Pyrophosphate / Q000378:metabolism; D017355:Zymomonas / Q000201:enzymology*
D001224:Aspartic Acid; D018698:Glutamic Acid; D011767:Pyruvate Decarboxylase; D013835:Thiamine Pyrophosphate