TY - JOUR
T1 - Analysis of hydrogen bonding in enzyme-substrate complexes of chloramphenicol acetyltransferase by infrared spectroscopy and site-directed mutagenesis
AU - Murray, Iain A.
AU - Derrick, Jeremy P.
AU - White, Andrew J.
AU - Drabble, Kevin
AU - Wharton, Christopher W.
AU - Shaw, William V.
PY - 1994
Y1 - 1994
N2 - Chloramphenicol acetyltransferase (CAT) reversibly transfers an acetyl group between CoA and the 3-hydroxyl of either chloramphenicol (Cm) or 1-acetylchloramphenicol (1AcCm). The products of the forward reactions, 3-acetylchloramphenicol (3-AcCm) and 1,3-diacetylchloramphenicol (1,3Ac2-Cm), are the substrates for the reverse reaction. The role of the 3-acetyl carbonyl group in the binding of the substrates 3AcCm and 1,3Ac2Cm to CAT has been investigated using infrared spectroscopy. Comparison of difference spectra (3-[12C=O]acetyl- minus 3-[13C=O]acetyl-) 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 carbonyl stretch in the wild-type complex, indicative of a hydrogen bond between this carbonyl and the hydroxyl group of Ser-148. The carbonyl bandwidth in the wild-type complex is reduced by 33% compared to that for the complex with S148A, indicating restriction of carbonyl mobility and dispersion in the former, an observation consistent with the proposed hydrogen bond between the ester carbonyl and the hydroxyl of Ser-148. Repetition of the experiment using 1,3Ac2Cm as the ligand reveals a frequency shift of only 3 cm-1 between wild-type and S148A complexes, indicating only a small change in the strength of carbonyl interaction. As the carbonyl stretch frequency (1727 cm-1) for the wild-type CAT complex with 1,3Ac2Cm is the same as that of the S148A enzyme complex with 3AcCm, it is likely that the hydrogen bond of the 3-acetyl carbonyl with Ser-148 is absent in the complex of CAT with 1,3Ac2Cm and therefore that (a) the modes of binding of the two ligands are different and (b) an explanation is available for the relative inefficiency of interconversion of 1-acetylchloramphenicol and 1,3 Ac2Cm. More generally, the results also illustrate the value of infrared spectroscopy in the study of the binding of alternative ligands to the same active site. © 1994 American Chemical Society.
AB - Chloramphenicol acetyltransferase (CAT) reversibly transfers an acetyl group between CoA and the 3-hydroxyl of either chloramphenicol (Cm) or 1-acetylchloramphenicol (1AcCm). The products of the forward reactions, 3-acetylchloramphenicol (3-AcCm) and 1,3-diacetylchloramphenicol (1,3Ac2-Cm), are the substrates for the reverse reaction. The role of the 3-acetyl carbonyl group in the binding of the substrates 3AcCm and 1,3Ac2Cm to CAT has been investigated using infrared spectroscopy. Comparison of difference spectra (3-[12C=O]acetyl- minus 3-[13C=O]acetyl-) 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 carbonyl stretch in the wild-type complex, indicative of a hydrogen bond between this carbonyl and the hydroxyl group of Ser-148. The carbonyl bandwidth in the wild-type complex is reduced by 33% compared to that for the complex with S148A, indicating restriction of carbonyl mobility and dispersion in the former, an observation consistent with the proposed hydrogen bond between the ester carbonyl and the hydroxyl of Ser-148. Repetition of the experiment using 1,3Ac2Cm as the ligand reveals a frequency shift of only 3 cm-1 between wild-type and S148A complexes, indicating only a small change in the strength of carbonyl interaction. As the carbonyl stretch frequency (1727 cm-1) for the wild-type CAT complex with 1,3Ac2Cm is the same as that of the S148A enzyme complex with 3AcCm, it is likely that the hydrogen bond of the 3-acetyl carbonyl with Ser-148 is absent in the complex of CAT with 1,3Ac2Cm and therefore that (a) the modes of binding of the two ligands are different and (b) an explanation is available for the relative inefficiency of interconversion of 1-acetylchloramphenicol and 1,3 Ac2Cm. More generally, the results also illustrate the value of infrared spectroscopy in the study of the binding of alternative ligands to the same active site. © 1994 American Chemical Society.
U2 - 10.1021/bi00199a003
DO - 10.1021/bi00199a003
M3 - Article
SN - 0006-2960
VL - 33
SP - 9826
EP - 9830
JO - Biochemistry
JF - Biochemistry
IS - 33
ER -