PROPERTIES OF HYBRID ACTIVE-SITES IN OLIGOMERIC PROTEINS - KINETIC AND LIGAND-BINDING STUDIES WITH CHLORAMPHENICOL ACETYLTRANSFERASE TRIMERS

Alteration of the charge of surface lysyl residues of chloramphenicol acetyltransferase (CAT) by site-directed mutagenesis was used to incre ase the: charge difference between the subunits of two naturally occur ring enzyme variants (CAT(I) and CAT(III)). The introduced charge chan ge greatly facilitates the purification of CAT(I)/CAT(III) and CAT(III )/CAT(III) hybrid trimers by ion-exchange chromatography. Hybrids cont aining only one functional active site per trimer were generated in vi tro by reversible denaturation of mixtures of ''active'' subunits (ret ention of a catalytic histidine at position 195) and ''inactive'' subu nits (with alanine replacing histidine 195). Such hybrids were used (1 ) to demonstrate that the previously observed novel binding of a stero idal antibiotic (fusidic acid) by CAT(I) involves amino acid residues at each subunit interface and (2) to identify specific residues contri buting to such interactions. A pre-steady-state kinetic characterizati on of homotrimers containing the H195A substitution also revealed that fusidate binding to CATI may. like chloramphenicol binding, involve a hydrogen bond with the catalytic histidine residue. In addition, conf irmation of the fact that His-195 interacts with chloramphenicol in CA T(I) as well as in CAT(III) makes it likely that it is essential for t he catalytic mechanism of all naturally occurring variants of CAT. as first suggested by structural evidence for the type III enzyme (Leslie , 1990).