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.