Abstract
The structure of the type III variant of chloramphenicol acetyltransferase reveals that Thr-174, a conserved residue, is hydrogen-bonded to a bound water molecule (water 252). Modeling studies (P. C. E. Moody and A. G. W. Leslie, unpublished data) suggested that water 252 could play a part in transition state stabilization via a hydrogen bond to the oxyanion of the putative tetrahedral intermediate. In addition, water 252 is one of three bound water molecules hydrogen-bonded to the 1-hydroxyl group of chloramphenicol in the chloramphenicol acetyltransferase-chloramphenicol binary complex. A combination of site-directed mutagenesis and the use of an alternative substrate has allowed the quantitation of the energetic contribution of each of the interactions made by water 252 to catalysis. Thr-174 was replaced by alanine, valine, and isoleucine, each substitution removing the hydroxyl group hydrogen-bonded to water 252. Steady-state kinetic analysis of the mutant enzymes was carried out using both chloramphenicol and 1-deoxy-chloramphenicol as acetyl acceptors. The substitutions at Thr-174 result in a fall in kcat and in decreased affinities for each acetyl acceptor in the binary complexes and also in the ternary complexes with acetyl-CoA. From the calculated free energies in the transition state, the hydrogen bond between water 252 and the oxyanion of the tetrahedral intermediate can be estimated to contribute 0.9 kcal mol-1 toward transition state stabilization, whereas the free energy of the hydrogen bonds between the 1-hydroxyl of chloramphenicol and three bound water molecules provides 1.6 kcal mol-1.
Highlights
The structure of the typeIII variant of chloramphen- ecules
An example of a water molecule amenable to this typeof icol acetyltransferase reveals that Thr-174, a conservedanalysis occurs with chloramphenicol acetyltransferase, residue, is hydrogen-bonded toa bound water molecule wherein one of the hydrogen bonds between the enzyme and
252 the role of this water molecule can be investicould play a part in transition state stabilization via a gated by alteration of enzyme or substrate
Summary
The three active sitesofCAT are located at each intertribute 0.9 kcal mol” toward transition state stabiliza- subunit interface, suchthat the catalytically essential imidaztion, whereas the free energy of the hydrogen bonds ole ring of His-195 is supplied from one subunit, whereas the between the 1-hydroxyl of chloramphenicol and three bound water molecules provides1.6 kcal mol-l. Fied in highly refined x-ray structures of enzymes may play A mechanism has been proposed for CAT wherein His-195 important roles in substrate binding and catalysis.Confirma- acts as a general base, abstractinagproton fromthe 3-hydroxyl tion of the functional importanceof such water molecules has of chloramphenicol, promoting nucleophilic attack on the tended to come from the conservation of bound water either in thioester of acetyl-coA. Bound Water in ll-ansition State Stabilizationby CAT bound water 252 contribute significantly to transitionstate stabilization via the two hydrogen bonding interactions proposed on the basisof the modeled tetrahedral intermediate
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