Fluorescence energy transfer in tryptophanase

Abstract

Excitation of apotryptophanase from Escherichia coli B lt7-A at 290 nm yielded a fluorescence emission centered at 340 nm. Binding of pyridoxal phosphate to apoenzyme induced quenching of protein fluorescence concomitant with an appearance of another peak at 510 nm by way of energy transfer from tryptophan. Based on the results, an approximate distance between the coenzyme and tryptophan was estimated to be 18–24 Å according to the Förster's theory. The ozone-inactivated enzyme yielded only the 340 nm-peak upon excitation at 290 nm following reconstitution with the coenzyme. The fluorescence decay time of the tryptophyl residue was somewhat increased by ozone-inactivation. These results suggest that the tryptophyl residue essential for the activity is involved in a direct interaction with the coenzyme.