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 mu tagenesis, has allowed the determination of the fluorescence lifetimes of each of the three tryptophan residues of the type III variant of c hloramphenicol acetyltransferase (CAT(III)) The mutant proteins retain ing a single tryptophan yield lifetimes of 1.36, 2.00, and 1.17 ns for Trp-16, -86, and -152, respectively. Binding of chloramphenicol short ens 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 di stances by energy-transfer calculations are in good agreement with tho se determined from the crystal structure of CAT(III). Despite binding at the same site in wild-type CAT(III), CoA and ethyl-S-CoA produce di fferent responses in global lifetime measurements-increases of 8% and 31%, respectively. Examination of each of the one-Trp CAT(III) variant s, generated by site-directed mutagenesis, yields a variety of respons es. Trp-152, located within the CoA binding site, responds to both CoA and its thioalkyl derivative with a 27-30% increase in fluorescence l ifetime. Trp-16, distant from the CoA site, does not differentiate bet ween the two ligands (7% increase in lifetime). However, Trp-86 shows a striking difference in binding responses, only a 4% decrease with Co A but a 14% reduction with ethyl-S-CoA. Each of the two-Trp CAT varian ts shows little change in global fluorescence lifetime on association with CoA. Although the binding of ethyl-S-CoA produces virtually no ch ange with the W152F variant, significant increases (25-33%) in fluores cence lifetime are observed with each of the two CAT(III) variants tha t retain Trp-152 together with either Trp-16 or Trp-86. The measuremen t of tryptophan lifetimes in two nonproductive ternary complexes allow ed the estimation of distances between tryptophan residues and Cm by c alculations based on energy transfer. The inferred increase (similar t o 6 Angstrom) in distance between Trp-86 and Cm in the ternary complex , compared to that in the binary (CAT . Cm) complex is large and unlik ely to be due solely to conformational changes, suggesting that change s in orientation factor and/or the flexibility or rotational freedom o f the indole side chain of Trp-86 may be contributory.