Properties of hybrid active sites in oligomeric proteins: kinetic and ligand binding studies with chloramphenicol acetyltransferase trimers.

Article Details

Citation

Copy to clipboard

Day PJ, Murray IA, Shaw WV

Properties of hybrid active sites in oligomeric proteins: kinetic and ligand binding studies with chloramphenicol acetyltransferase trimers.

Biochemistry. 1995 May 16;34(19):6416-22.

PubMed ID

7756272 [ View in PubMed

]

Abstract

Alteration of the charge of surface lysyl residues of chloramphenicol acetyltransferase (CAT) by site-directed mutagenesis was used to increase the charge difference between the subunits of two naturally occurring enzyme variants (CATI and CATIII). The introduced charge change greatly facilitates the purification of CATI/CATIII and CATIII/CATIII hybrid trimers by ion-exchange chromatography. Hybrids containing only one functional active site per trimer were generated in vitro by reversible denaturation of mixtures of "active" subunits (retention of a catalytic histidine at position 195) and "inactive" subunits (with alanine replacing histidine 195). Such hybrids were used (1) to demonstrate that the previously observed novel binding of a steroidal antibiotic (fusidic acid) by CATI involves amino acid residues at each subunit interface and (2) to identify specific residues contributing to such interactions. A pre-steady-state kinetic characterization 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, confirmation of the fact that His-195 interacts with chloramphenicol in CATI as well as in CATIII makes it likely that it is essential for the catalytic mechanism of all naturally occurring variants of CAT, as first suggested by structural evidence for the type III enzyme (Leslie, 1990).

DrugBank Data that Cites this Article

Drug Targets

Drug	Target	Kind	Organism	Pharmacological Action	Actions
Fusidic acid	Chloramphenicol acetyltransferase	Protein	Salmonella typhi	Unknown	Inhibitor	Details
Fusidic acid	Chloramphenicol acetyltransferase 3	Protein	Escherichia coli	Unknown	Inhibitor	Details