Stabilization of the imidazole ring of His-195 at the active site of chloramphenicol acetyltransferase

The imidazole of His-195 plays an essential role in the proposed general base mechanism of chloramphenicol acetyltransferase (CAT). The structure of the binary complex of CATIII and chloramphenicol suggests that two unusual interactions might determine the conformation of the side chain of His-195: (i) an intraresidue hydrogen bond between its main chain carbonyl and the protonated N delta 1 of the imidazole ring and (ii) face-to-face van der Waals contact between the His-195 imidazole group and the aromatic side chain of Tyr-25. Tyr-25 also makes a hydrogen bond, via its phenolic hydroxyl, to the carbonyl oxygen of the substrate chloramphenicol. Replacement of Tyr-25 of CATIII by phenylalanine results in a modest increase in the Km for chloramphenicol (from 11.6 to 14.6 microM) and a 2-fold fall in kcat (599 to 258 s-1), indicative of a free energy contribution to transition state binding of 0.6 kcal mol-1 for the hydrogen bond between Tyr-25 and chloramphenicol. In contrast, substitution of Tyr-25 by alanine yields an enzyme that is dramatically impaired in its ability to bind chloramphenicol (Km = 173 microM). As kcat for Ala-25 CAT is also reduced (130 s-1), the loss of the aryl group results in a 69-fold decrease in kcat/Km, corresponding to a free energy contribution to binding and catalysis of 2.5 kcal mol-1. In addition to the loss of the hydrogen bond between Tyr-25 and chloramphenicol, the loss of substrate affinity in Ala-25 CAT may be a direct consequence of reduced hydrophobicity of the chloramphenicol-binding site and/or the loss of critical constraints on the precise conformation of the catalytic imidazole. However, as with wild type CAT, inactivation of Ala-25 CAT by the affinity reagent 3-(bromoacetyl) chloramphenicol is accompanied by modification solely at N epsilon 2 of His-195. Hence, the results demonstrate that tautomeric stabilization of the imidazole ring persists in the absence of van der Waals interactions with the side chain of Tyr-25, probably as a consequence of hydrogen bonding between the protonated N delta 1 and the carbonyl oxygen of His-195.