Probing the role of the C-terminus of Bacillus subtilis chorismate mutase by a novel random protein-termination strategy

A novel strategy combining random protein truncation and genetic selection has been developed to identify dispensable C-terminal segments of an enzyme . This approach, which entails the random introduction of premature termina tion codons, was applied to the last 17 residues of chorismate mutase from Bacillus subtilis (BsCM). Although structurally ill-defined, the C-terminus of BsCM has been proposed to cap the active site upon substrate binding an d affect catalysis, However, sequence patterns of 178 selected gene variant s show that the final 11 residues of the protein can be mutated and even re moved without significantly impairing activity in vivo. In fact, none of th e randomized residues is absolutely required, but a preference for wild-typ e Lys111, Ala112, Leu115, and Arg116 is apparent. These residues are part o f a C-terminal 3(10)-helix and provide contacts with the rest of the protei n or its ligands, The kinetic parameters of selected enzyme variants show t hat truncations and mutations do not significantly impair catalytic turnove r (k(cat)) but substantially decrease k(cat)/K-m. Thus, while the 17 C-term inal residues of BsCM do not participate directly in the chemical rearrange ment, they appear to contribute to enzymatic efficiency via uniform binding of the substrate and transition state.