The role of residues outside the active site: structural basis for function of C191 mutants of Escherichia coli aspartate aminotransferase

In previous kinetic studies of Escherichia coli aspartate aminotransferase, it was determined that some substitutions of conserved cysteine 191, which is located outside of the active site, altered the kinetic parameters of t he enzyme (Gloss,L.M., Spencer,D.E. and Kirsch,J.F., 1996, Protein Struct. Funct. Genet., 24, 195-208). The mutations resulted in an alkaline shift of 0.6-0.8 pH units for the pK(a) of the internal aldimine between the PLP co factor and Lys258, The change in the pK(a) affected the pH dependence of th e k(cat)/K-m (aspartate) values for the mutant enzymes. To help to understa nd these observations, crystal structures of five mutant forms of E,coli as partate aminotransferase (the maleate complexes of C191S, C191F, C191Y and C191W, and C191S without maleate) were determined at about 2 Angstrom resol ution in the presence of the pyridoxal phosphate cofactor, The overall thre e-dimensional fold of each mutant enzyme is the same as that of the wild-ty pe protein, but there is a rotation of the mutated side chain around its C- alpha-C-beta bond. This side chain rotation results in a change in the patt ern of hydrogen bonding connecting the mutant residue and the protonated Sc hiff base of the cofactor, which could account for the altered pK(a) of the Schiff base imine nitrogen that was reported previously, These results dem onstrate how residues outside the active site can be important in helping d etermine the subtleties of the active site amino acid geometries and intera ctions and how mutations outside the active site can have effects on cataly sis, In addition, these results help explain the surprising result previous ly reported that, for some mutant proteins, replacement of a buried cystein e with an aromatic side chain did not destabilize the protein fold. Instead , rotation around the C-alpha-C-beta bond allowed each large aromatic side chain to become buried in a nearby pocket without large changes in the enzy me's backbone geometry.