Leucine-160 of chloramphenicol acetyltransferase (CAT) has been replaced by site-directed mutagenesis to investigate enzyme-ligand interactions at the 1-hydroxyl substituent of the substrate chloramphenicol. The consequences of the substitution of Leu-160 by glutamine and by phenylalanine were deduced from the steady-state kinetic parameters for acetyl transfer from acetyl-CoA to the 3-hydroxyl of chloramphenicol and its analogues 1-deoxychloramphenicol and 1-acetylchloramphenicol. The acetyl group of the latter, which is a substrate both in vivo and in vitro, could potentially bind in a similar position to the 1-hydroxyl of chloramphenicol, in close proximity to the side chain of Leu-160. In the case of Gln-160 CAT, large increases in Km for the three acetyl acceptors were accompanied by small decreases in kcat and in apparent affinity for acetyl-CoA. Such results are consistent with the introduction of the relatively hydrophilic amide in place of the delta-methyl groups of Leu-160. The kinetic properties of Phe-160 CAT were unexpected in that Km for each of the three acetyl acceptors was unchanged or reduced, compared to the equivalent parameters for the wild-type enzyme, whereas kcat fell significantly (44-83-fold) in each case. The ratios of specificity constants (kcat/Km) for the acetylation of chloramphenicol compared with the alternative acyl acceptors were similar for wild-type and mutant enzymes. As the residue substitutions for Leu-160 do not result in enhanced discrimination against the binding and acetylation of 1-acetylchloramphenicol, it appears unlikely that the 1-acetyl group binds to the CAT active site in the same position as that occupied by the 1-hydroxyl of chloramphenicol.(ABSTRACT TRUNCATED AT 250 WORDS)