Substrate binding to chloramphenicol acetyltransferase: evidence for negative cooperativity from equilibrium and kinetic constants for binary and ternary complexes.
Chloramphenicol acetyltransferase (CAT) catalyzes the acetyl-CoA-dependent acetylation of chloramphenicol by a ternary complex mechanism with a rapid equilibrium and essentially random order of addition of substrates. Such a kinetic mechanism for a two-substrate reaction provides an opportunity to compare the affinity of enzyme for each substrate in the binary complexes (1/Kd) with corresponding values (1/Km) for affinities in the ternary complex where any effect of the other substrate should be manifest. The pursuit of such information for CAT involved the use of four independent methods to determine the dissociation constant (Kd) for chloramphenicol in the binary complex, techniques which included stopped-flow measurements of on and off rates, and a novel fluorometric titration method. The binary complex dissociation constant (Kd) for acetyl-CoA was measured by fluorescence enhancement and steady-state kinetic analysis. The ternary complex dissociation constant (Km) for each substrate (in the presence of the other) was determined by kinetic and fluorometric methods, using CoA or ethyl-CoA to form nonproductive ternary complexes. The results demonstrate an unequivocal decrease in affinity of CAT for each of its substrates on progression from the binary to the ternary complex, a phenomenon most economically described as negative cooperativity. The binary complex dissociation constants (Kd) for chloramphenicol and acetyl-CoA are 4 microM and 30 microM whereas the corresponding dissociation constants in the ternary complex (Km) are 12 microM and 90 microM, respectively.(ABSTRACT TRUNCATED AT 250 WORDS)
Department of Biochemistry, University of Leicester, U.K.
J
J
Ellis
C R
CR
Bagshaw
W V
WV
Shaw
D000105:Acetyl Coenzyme A / Q000378:metabolism*; D001667:Binding, Competitive; D002701:Chloramphenicol / Q000378:metabolism*; D015500:Chloramphenicol O-Acetyltransferase / Q000378:metabolism*; D004926:Escherichia coli / Q000201:enzymology*; D005453:Fluorescence; D007700:Kinetics; D012044:Regression Analysis; D013050:Spectrometry, Fluorescence; D014364:Tryptophan
D002701:Chloramphenicol; D000105:Acetyl Coenzyme A; D014364:Tryptophan; D015500:Chloramphenicol O-Acetyltransferase