Functional and structural analysis of cis-proline mutants of Escherichia coli aspartate aminotransferase

TO elucidate the role of the two conserved cis-proline residues of aspartat e aminotransferase (AspAT), one double and two single mutants of the enzyme from Escherichia coli (EcAspAT) were prepared: P138A, P195A and P138A/P195 A in which the two prolines were replaced by alanine. The crystal structure s of P195A and P138A/P195A have been determined at 2.3-2.1 Angstrom resolut ion. The wild-type geometry, including the cis conformation of the 194-195 peptide bond is retained upon substitution of proline 195 by alanine, where as the trans conformation is adopted at the 137-138 peptide bond. Quite sur prisingly, the replacement of each of the two prolines by alanine does not significantly affect either the activity or the stability of the protein. A ll the three mutants follow the same pathway as the wild type for unfolding equilibrium induced by guanidine hydrochloride [Herold, M., and Kirschner, K. (1990) Biochemistry 29, 1907-1913]. The kinetics of renaturation of P19 5A, where the alanine retains the wild-type cis conformation, is faster tha n wild type, whereas renaturation of P138A, which adopts the trans conforma tion, is slower. We conclude that cis-prolines seem to have been retained t hroughout the evolution of aspartate aminotransferase to possibly play a su btle role in directing the traffic of intermediates toward the unique struc ture of the native state, rather than to respond to the needs for a specifi c catalytic or functional role.