MODULAR MUTAGENESIS OF A TIM-BARREL ENZYME - THE CRYSTAL-STRUCTURE OFA CHIMERIC ESCHERICHIA-COLI TIM HAVING THE 8TH BETA-ALPHA-UNIT REPLACED BY THE EQUIVALENT UNIT OF CHICKEN TIM

The crystal structure of a hybrid Escherichia coli triosephosphate iso merase (TIM) has been determined at 2.8 Angstrom resolution. The hybri d TIM (ETIM8CHI) was constructed by replacing the eighth beta alpha-un it of E. coli TIM with the equivalent unit of chicken TIM. This replac ement involves 10 sequence changes. One of the changes concerns the mu tation of a buried alanine (Ala232 in strand 8) into a phenylalanine. The ETIM8CHI structure shows that the A232F sequence change can be inc orporated by a side-chain rotation of Phe224 (in helix 7). No cavities or strained dihedrals are observed in ETIM8CHI in the region near pos ition 232, which is in agreement with the observation that ETIM8CHI an d E.coli TIM have similar stabilities. The largest CA (C-alpha atom) m ovements, similar to 3 Angstrom, are seen for the C-terminal end of he lix 8 (associated with the outward rotation of Phe224) and for the res idues in the loop after helix 1 (associated with the sequence changes in helix 8). From the structure it is not clear why the k(cat) of ETIM 8CHI is 10 times lower than in wild type E.coli TIM.