TRANSITION-STATE STABILIZATION BY CHLORAMPHENICOL ACETYLTRANSFERASE -ROLE OF A WATER MOLECULE BOUND TO THREONINE-174

The structure of the type III variant of chloramphenicol acetyltransfe rase reveals that Thr-174, a conserved residue, is hydrogen-bonded to a bound water molecule (water 252). Modeling studies (P. C. E. Moody a nd A. G. W. Leslie, unpublished data) suggested that water 252 could p lay a part in transition state stabilization via a hydrogen bond to th e oxyanion of the putative tetrahedral intermediate. In addition, wate r 252 is one of three bound water molecules hydrogen-bonded to the 1-h ydroxyl group of chloramphenicol in the chloramphenicol acetyltransfer ase-chloramphenicol binary complex. A combination of site-directed mut agenesis and the use of an alternative substrate has allowed the quant itation of the energetic contribution of each of the interactions made by water 252 to catalysis. Thr-174 was replaced by alanine, valine, a nd isoleucine, each substitution removing the hydroxyl group hydrogen- bonded to water 252. Steady-state kinetic analysis of the mutant enzym es was carried out using both chloramphenicol and 1-deoxy-chlorampheni col as acetyl acceptors. The substitutions at Thr-174 result in a fall in k(cat) and in decreased affinities for each acetyl acceptor in the binary complexes and also in the ternary complexes with acetyl-CoA. F rom the calculated free energies in the transition state, the hydrogen bond between water 252 and the oxyanion of the tetrahedral intermedia te can be estimated to contribute 0.9 kcal mol-1 toward transition sta te stabilization, whereas the free energy of the hydrogen bonds betwee n the 1-hydroxyl of chloramphenicol and three bound water molecules pr ovides 1.6 kcal mol-1.