Analysis of Active Site Residues in Escherichia coli Chorismate Mutase by Site-Directed Metagenesis

摘要

The X-ray crystal structures of three proteins that catalyze the Claisen rearrangement of chorismate (1) to prephenate (2) have been solved as complexes with the endo-oxabicyclic transition state analogue 3 (Scheme 1). Analysis of the structures of the chorismate mutase from Bacillus subtilis, the N-terminal 109 amino acid catalytic fragment of the bifunctional Escherichia coli choiismate mutase-ptephenate dehydratase ("P protein"), and the catalytic antibody 1F7 indicate that the active sites of both enzyme and antibody mutases are complementary to the conformationally restricted transition state analogue 3. In the case, of the enzymes, the structures also reveal a number of groups that could function in the formation or stabilization of a polar transition state generated by the heterolysis of the O7-C5 bond. These structures, taken together with earlier studies and recent mutational analyses of active site residues, provide a unique opportunity to identify common mechanistic features associated with this novel biological transformation. We describe here the generation and characterization of 13 active site mutants of the E coli monofunctional mutase (EcCM, Figure 1) and compare the properties of these mutants with the analogous mutants of the B. subtilis enzyme (BsCM).