Transition state stabilization by chloramphenicol acetyltransferase. Role of a water molecule bound to threonine 174.
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 Modeling studies (P. C. E. Moody and A. G. W. Leslie, unpublished data) suggested that water 252 could play a part in transition state stabilization via a hydrogen bond to the oxyanion of the putative tetrahedral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.
Lewendon
A
A
Department of Biochemistry, University of Leicester, United Kingdom.
Shaw
W V
WV
D001483:Base Sequence; D001665:Binding Sites; D002384:Catalysis; D015500:Chloramphenicol O-Acetyltransferase / Q000737:chemistry / Q000378:metabolism*; D004795:Enzyme Stability; D004926:Escherichia coli; D006860:Hydrogen Bonding; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D009838:Oligodeoxyribonucleotides; D013912:Threonine / Q000737:chemistry / Q000378:metabolism*; D014867:Water / Q000737:chemistry / Q000378:metabolism*
D009838:Oligodeoxyribonucleotides; D014867:Water; D013912:Threonine; D015500:Chloramphenicol O-Acetyltransferase