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 act ive site is inaccessible to solvent during catalysis. However, the carboxy/ 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 carb oxyl terminus by making 15 variants of the enzyme with serial deletions. Th e activity is largely unaffected by removal of up to seven residues but del etion 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 be tween pyruvate and larger substrates, and in cofactor affinity. Several sub stitution mutants at residues R561 and S560 were prepared, purified, and fu lly characterized. The results indicate important roles for the side-chain of R561 and the backbone atoms of S560. It is suggested that the carboxyl-t erminal region of pyruvate decarboxylase is needed to lock in the cofactors and for the proper closure of the active site that is required for discrim ination between substrates and for decarboxylation to occur.