Replacement of lysine 234 affects transition state stabilization in the active site of beta-lactamase TEM1.

摘要: Lysine 234 is a residue highly conserved in all beta-lactamases, except the carbenicillin-hydrolyzing enzymes, which it replaced by an arginine. Informational suppression has been used to create amino acid substitutions at this position broad spectrum Escherichia coli beta-lactamase TEM-1, order elucidate role of lies on wall closed end active site cavity. The mutants K234R and K234T were constructed their kinetic constants measured. Replacement lysine arginine yields enzyme with similar activity toward cephalosporins most penicillins, carboxypenicillins for presence guanidine group enhances transition state binding. removal basic mutant protein variant retains low but losts drastically its ability hydrolyze cephalosporins. Moreover, these two mutations largely decreased affinity penicillins (10-fold 50-fold K234T). This can be correlated disruption predicted electrostatic binding between C3 carboxylic amine function lysine. Therefore, E. TEM-1 involved both initial recognition substrate stabilization.