Tryptophan fluorescence of chloramphenicol acetyltransferase: resolution of individual excited-state lifetimes by site-directed mutagenesis and multifrequency phase fluorometry.
Multifrequency phase fluorometry, in conjunction with site-directed mutagenesis, has allowed the determination of the fluorescence lifetimes of each of the three tryptophan residues of the type III variant of chloramphenicol acetyltransferase (CATIII). The mutant proteins retaining a single tryptophan yield lifetimes of 1.36, 2.00, and 1.17 ns for Trp-16, -86, and -152, respectively. Binding of chloramphenicol shortens the fluorescence lifetimes of all three tryptophans to some extent, in particular those of Trp-86 and Trp-152 (decreases of 51% and 39%, respectively). The mechanism of fluorescence quenching is believed to be radiationless energy transfer. Estimates of Trp-chloramphenicol distances by energy-transfer calculations are in good agreement with those determined from the crystal structure of CATIII. Despite binding at the same site in wild-type CATIII, CoA and ethyl-S-CoA produce different responses in global lifetime measurements--increases of 8% and 31%, respectively. Examination of each of the one-Trp CATIII variants, generated by site-directed mutagenesis, yields a variety of responses. Trp-152, located within the CoA binding site, responds to both CoA and its thioalkyl derivative with a 27-30% increase in fluorescence lifetime. Trp-16, distant from the CoA site, does not differentiate between the two ligands (7% increase in lifetime). However, Trp-86 shows a striking difference in binding responses, only a 4% decrease with CoA but a 14% reduction with ethyl-S-CoA. Each of the two-Trp CAT variants shows little change in global fluorescence lifetime on association with CoA.(ABSTRACT TRUNCATED AT 250 WORDS)
Ellis
J
J
Department of Biochemistry, University of Leicester, United Kingdom.
Bagshaw
C R
CR
Shaw
W V
WV
D015500:Chloramphenicol O-Acetyltransferase / Q000737:chemistry* / Q000235:genetics / Q000302:isolation & purification; D005453:Fluorescence; D005470:Fluorometry / Q000379:methods*; D016297:Mutagenesis, Site-Directed; D014364:Tryptophan / Q000737:chemistry*
D014364:Tryptophan; D015500:Chloramphenicol O-Acetyltransferase