FLUORESCENCE LIFETIMES OF THE TRYPTOPHAN RESIDUES IN ORNITHINE TRANSCARBAMOYLASE

Multifrequency (2-230 MHz) phase-modulation fluorescence measurements and site-directed mutagenesis have been employed to assign fluorescenc e lifetimes, quantum yields, and emission maxima to the four tryptopha ns in the enzyme ornithine transcarbamoylase from Escherichia coli (OT Case) (Trp-125, -192, -233, and -243). OTCase displays two apparent fl uorescence lifetimes, 7.2 and 3.2 ns. Results on specific mutants show that Trp-233 has a lifetime of 7.1 ns, while Trp- 125, -192, and -243 have lifetimes of 4.0, 3.6, and 4.9 ns, respectively. Thus, the speci fic conformational changes of the polypeptide segment involving Trp-23 3 may be monitored conveniently in the wild-type enzyme. On the basis of quantum yield values, Trp-233 is calculated to contribute approxima tely 43% of the fluorescence intensity of the enzyme, while direct mea surements of the enzyme show that up to 65% of the total intensity is really emitted by this tryptophan. The discrepancy may arise from ener gy transfer from Trp-125 to Trp-233, with an efficiency of 20%. Applic ation of the assigned tryptophan lifetimes to probe ligand-induced pro tein conformational changes has also been demonstrated.