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) 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.(ABSTRACT TRUNCATED AT 250 WORDS)
Murray
I A
IA
Department of Biochemistry, University of Leicester, United Kingdom.
Derrick
J P
JP
White
A J
AJ
Drabble
K
K
Wharton
C W
CW
Shaw
W V
WV
D000107:Acetylation; D001665:Binding Sites; D002701:Chloramphenicol / Q000031:analogs & derivatives / Q000378:metabolism; D015500:Chloramphenicol O-Acetyltransferase / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D003065:Coenzyme A / Q000378:metabolism; D004926:Escherichia coli / Q000235:genetics; D006860:Hydrogen Bonding; D016297:Mutagenesis, Site-Directed; D011994:Recombinant Proteins; D013055:Spectrophotometry, Infrared; D013329:Structure-Activity Relationship
D011994:Recombinant Proteins; C026822:chloramphenicol 3-acetate; C076648:1,3-diacetylchloramphenicol; C026821:chloramphenicol 1-acetate; D002701:Chloramphenicol; D015500:Chloramphenicol O-Acetyltransferase; D003065:Coenzyme A