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 (CAT(III)) 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 CAT(III). Despite binding at the same site in wild-type CAT(III), 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 CAT(III) 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. Although the binding of ethyl-S-CoA produces virtually no change with the W152F variant, significant increases (25-33%) in fluorescence lifetime are observed with each of the two CAT(III) variants that retain Trp-152 together with either Trp-16 or Trp-86. The measurement of tryptophan lifetimes in two nonproductive ternary complexes allowed the estimation of distances between tryptophan residues and Cm by calculations based on energy transfer. The inferred increase (similar to 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 unlikely to be due solely to conformational changes, suggesting that changes in orientation factor and/or the flexibility or rotational freedom of the indole side chain of Trp-86 may be contributory.