Effects of deletions at the carboxyl terminus of Zymomonas mobilis pyruvate decarboxylase on the kinetic properties and substrate specificity.
The three-dimensional structure of Zymomonas mobilis pyruvate decarboxylase shows that the carboxyl-terminal region of the protein occludes the active site. This observation is consistent with earlier suggestions that the active site is inaccessible to solvent during catalysis. However, the carboxyl-terminal region must move aside to allow entry of the substrate, and again to permit the products to leave. Here we have examined the role of the carboxyl terminus by making 15 variants of the enzyme with serial deletions. The activity is largely unaffected by removal of up to seven residues but deletion of the next two, R561 and S560, results in a drastic loss of activity. Five of these deletion mutants were purified and fully characterized and showed progressive decreases in activity, in the ability to discriminate between pyruvate and larger substrates, and in cofactor affinity. Several substitution mutants at residues R561 and S560 were prepared, purified, and fully characterized. The results indicate important roles for the side-chain of R561 and the backbone atoms of S560. It is suggested that the carboxyl-terminal region of pyruvate decarboxylase is needed to lock in the cofactors and for the proper closure of the active site that is required for discrimination between substrates and for decarboxylation to occur.
Chang
A K
AK
Centre for Protein Structure, Function and Engineering, Department of Biochemistry, The University of Queensland, Brisbane, Australia.
Nixon
P F
PF
Duggleby
R G
RG
D000595:Amino Acid Sequence; D019943:Amino Acid Substitution / Q000235:genetics; D001120:Arginine / Q000235:genetics; D001665:Binding Sites / Q000235:genetics; D003067:Coenzymes / Q000378:metabolism; D004789:Enzyme Activation / Q000235:genetics; D007700:Kinetics; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D010446:Peptide Fragments / Q000235:genetics* / Q000378:metabolism*; D011767:Pyruvate Decarboxylase / Q000235:genetics* / Q000378:metabolism*; D017384:Sequence Deletion; D012694:Serine / Q000235:genetics; D013379:Substrate Specificity / Q000235:genetics; D017355:Zymomonas / Q000201:enzymology* / Q000378:metabolism*
D003067:Coenzymes; D010446:Peptide Fragments; D012694:Serine; D001120:Arginine; D011767:Pyruvate Decarboxylase