ANALYSIS OF HYDROGEN-BONDING IN ENZYME-SUBSTRATE COMPLEXES OF CHLORAMPHENICOL ACETYLTRANSFERASE BY INFRARED-SPECTROSCOPY AND SITE-DIRECTED MUTAGENESIS

Chloramphenicol acetyltransferase (CAT) reversibly transfers an acetyl group between CoA and the 3-hydroxyl of either chloramphenicol (Cm) o r 1-acetylchloramphenicol (1AcCm). The products of the forward reactio ns, 3-acetylchloramphenicol (3-AcCm) and 1,3-diacetylchloramphenicol ( 1,3Ac(2)-Cm), are the substrates for the reverse reaction. The role of the 3-acetyl carbonyl group in the binding of the substrates 3AcCm an d 1,3Ac(2)Cm to CAT has been investigated using infrared spectroscopy. Comparison of difference spectra (3-[C-12=O]acetyl- minus 3-[C-13=O]a cetyl-) obtained for the binary complexes of 3AcCm with wild-type CAT, and with a variant wherein serine-148 is replaced by alanine (S148A), reveals a large (9 cm(-1)) down frequency shift for the 3-acetyl carb onyl stretch in the wild-type complex, indicative of a hydrogen bond b etween this carbonyl and the hydroxyl group of Ser-148. The carbonyl b andwidth in the wild-type complex is reduced by 33% compared to that f or the complex with S148A, indicating restriction of carbonyl mobility and dispersion in the former, an observation consistent with the prop osed hydrogen bond between the ester carbonyl and the hydroxyl of Ser- 148. Repetition of the experiment using 1,3Ac(2)Cm as the ligand revea ls a frequency shift of only 3 cm(-1) between wild-type and S148A comp lexes, indicating only a small change in the strength of carbonyl inte raction. As the carbonyl stretch frequency (1727 cm(-1)) for the wild- type CAT complex with 1,3Ac(2)Cm is the same as that of the S148A enzy me complex with 3AcCm, it is likely that the hydrogen bond of the 3-ac etyl carbonyl with Ser-148 is absent in the complex of CAT with 1,3Ac( 2)Cm and therefore that (a) the modes of binding of the two ligands ar e different and (b) an explanation is available for the relative ineff iciency of interconversion of 1-acetylchloramphenicol and 1,3Ac(2)Cm. More generally, the results also illustrate the value of infrared spec troscopy in the study of the binding of alternative ligands to the sam e active site.