Acetyl coenzyme A binding by chloramphenicol acetyltransferase: long-range electrostatic determinants of coenzyme A recognition.
The possible involvement of arginyl and lysyl side chains of chloramphenicol acetyltransferase (CAT) in binding coenzyme A (CoA) was studied by means of chemical modification, site-directed mutagenesis, variation in ionic strength, use of competitive inhibitors or substrate analogues, and X-ray crystallography. Unlike a number of enzymes, including citrate synthase, CAT does not employ specific ion pairs with the phosphoanionic centers of CoA to bind the acetyl donor, and arginyl residues play no role in recognition of the coenzyme. Although phenylglyoxal inactivates CAT reversibly, it does so by the formation of an unstable adduct with a thiol group, that of Cys-31 in the chloramphenicol binding site. The inhibitory effect of increasing ionic strength on kcat/Km(acetyl-CoA) can be explained by long-range electrostatic interactions between CoA and the epsilon-amino groups of Lys-54 and Lys-177, both of which are solvent-accessible. The epsilon-amino group of Lys-54 contributes 1.3 kcal.mol-1 to the binding of acetyl-CoA via interactions with both the 3'- and 5'-phosphoanions of CoA. Lys-177 contributes only 0.4 kcal.mol-1 to the productive binding of acetyl-CoA, mediated by long-range (approximately 14 A) interactions with the 5'-alpha- and -beta-phosphoanions of CoA. The combined energetic contribution of Lys-54 and Lys-177 to acetyl-CoA binding (1.7 kcal.mol-1) is less than that previously demonstrated (2.4 kcal.mol-1) for a simple hydrophobic interaction between Tyr-178 and the adenine ring of CoA (Day & Shaw, 1992).(ABSTRACT TRUNCATED AT 250 WORDS)
Day
P J
PJ
Department of Biochemistry, University of Leicester, U.K.
Shaw
W V
WV
Gibbs
M R
MR
Leslie
A G
AG
D000105:Acetyl Coenzyme A / Q000737:chemistry / Q000378:metabolism*; D001120:Arginine / Q000737:chemistry; D001665:Binding Sites; D015500:Chloramphenicol O-Acetyltransferase / Q000037:antagonists & inhibitors / Q000737:chemistry / Q000235:genetics*; D004926:Escherichia coli / Q000201:enzymology; D007700:Kinetics; D008239:Lysine / Q000737:chemistry; D016297:Mutagenesis, Site-Directed; D009994:Osmolar Concentration; D010658:Phenylglyoxal / Q000737:chemistry; D013329:Structure-Activity Relationship
D000105:Acetyl Coenzyme A; D001120:Arginine; D015500:Chloramphenicol O-Acetyltransferase; D008239:Lysine; D010658:Phenylglyoxal