Fluorescence energy transfer studies on the active site of papain.
Measurements have been performed of the excited-state lifetimes and fluorescence yields of papain tryptophan units when acyl derivatives of Phe-glycinal are bound at the active site of the enzyme. The enhancement of tryptophan fluorescence in complexes of papain with the acetyl or benzyloxycarbonyl derivatives is not stereospecific with respect to the configuration of the phenylalanyl residue, and the L and D isomers are equally effective as active-site-directed inhibitors of papain action. Evidence is offered in favor of the conclusion that this enhancement is primarily a consequence of the interaction of the phenylalanyl side chain of the inhibitor with Trp-69 of the enzyme. This residue can exchange fluorescence energy with the other four tryptophans of papain (Trp-7, Trp-26, Trp-177, Trp-181) upon excitation near their absorption maxima, but such "homotransfer" is absent if they are excited at the long-wave edge of their absorption spectra. Crystallographic data indicate that Trp-26 is most favorably positioned for efficient energy exchange with Trp-69, and the fluorescence data have been used to calculate a distance of 11 A between the two residues; this value is in satisfactory agreement with that found by crystallography. When derivatives of Phe-glycinal bearing an amino-terminal mansyl [6-(N-methylanilino)-2-naphthalene sulfonyl] group are bound at the active site of papain, the tryptophan fluorescence is quenched, as compared with that of the complex of papain with acetyl-Phe-glycinal, indicating energy transfer from papain tryptophan (most probably via Trp-26) to the fluorescent probe group. Although the L and D isomers of mansyl-Phe-glycinal are equally effective as inhibitors of papain action, the fluorescence quenching by the two isomers is different.
D001665:Binding Sites; D004735:Energy Transfer; D008024:Ligands; D009842:Oligopeptides / Q000378:metabolism / Q000494:pharmacology; D010206:Papain / Q000037:antagonists & inhibitors; D010649:Phenylalanine; D011487:Protein Conformation; D013050:Spectrometry, Fluorescence; D013379:Substrate Specificity; D014364:Tryptophan
D008024:Ligands; D009842:Oligopeptides; D010649:Phenylalanine; D014364:Tryptophan; D010206:Papain