Acetyl coenzyme A binding by chloramphenicol acetyltransferase. Hydrophobic determinants of recognition and catalysis.
The preponderance of nonpolar contacts between CoA and chloramphenicol acetyltransferase in the high resolution structure of the binary complex prompted a study of selected hydrophobic residues by site-directed mutagenesis and steady-state kinetic analysis. Substitutions of three aromatic residues were used to evaluate binding contacts with the adenine moiety of CoA (Tyr-178), the pantetheine arm of the coenzyme (Tyr-56), and the S-acyl substituent (Phe-33). For those substitutions at residues 56 and 178 that cannot promote alternative polar interactions there is a correlation between residue hydrophobicity and the free energy of formation of the binary and ternary complexes of acetyl-CoA and chloramphenicol acetyltransferase and of the transition-state complex. Substitutions at Tyr-178 destabilize all such complexes to approximately the same extent (uniform binding changes), whereas those at Tyr-56 and Phe-33 cause differential binding changes, having a greater effect on the transition state than on either of the other complexes with acetyl-CoA.
Day
P J
PJ
Department of Biochemistry, University of Leicester, United Kingdom.
Shaw
W V
WV
D000105:Acetyl Coenzyme A / Q000378:metabolism*; D000595:Amino Acid Sequence; D001665:Binding Sites; D015500:Chloramphenicol O-Acetyltransferase / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D004795:Enzyme Stability; D004926:Escherichia coli / Q000235:genetics; D007700:Kinetics; D008958:Models, Molecular; D016297:Mutagenesis, Site-Directed; D011487:Protein Conformation; D011994:Recombinant Proteins / Q000737:chemistry / Q000302:isolation & purification / Q000378:metabolism; D013816:Thermodynamics
D011994:Recombinant Proteins; D000105:Acetyl Coenzyme A; D015500:Chloramphenicol O-Acetyltransferase