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TRYPTOPHANYL FLUORESCENCE QUENCHING OF UROCANASE FROM Pseudomonas putida BY ACRYLAMIDE, CESIUM, IODIDE, and IMIDAZOLEPROPIONATE
Author(s) -
O'Donneix Peter S.,
Hug Daniel H.
Publication year - 1985
Publication title -
photochemistry and photobiology
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 0.818
H-Index - 131
eISSN - 1751-1097
pISSN - 0031-8655
DOI - 10.1111/j.1751-1097.1985.tb01546.x
Subject(s) - chemistry , tryptophan , iodide , quenching (fluorescence) , acrylamide , fluorescence , photochemistry , inorganic chemistry , biochemistry , organic chemistry , amino acid , physics , quantum mechanics , copolymer , polymer
— Urocanase from Pseudomonas putida can be photoactivated by UV radiation. Because of the action spectrum peak at 280 nm, tryptophan has been implicated in the photoactivation by energy transfer. In these studies, tryptophan was determined, the exposure and environment of tryptophanyl residues were studied with collisional quenchers, and the involvement of tryptophanyl residues in the photoactivation of urocanase was investigated. There are sixteen tryptophanyl residues per urocanase molecule as measured by two methods. Fluorescence quenching with acrylamide, cesium, and iodide was used to describe the accessibility and environment of urocanase tryptophanyl residues. Quenching constants indicated there is little difference in the accessibility of tryptophanyl residues between active and inactive enzyme. Tryptophanyl residues of native enzyme were most accessible to acrylamide ( f a , = 0.6–0.7), less accessible to iodide ( f a = 0.4), and not accessible to cesium ion, suggesting that surface residues were in regions of positive charge. Stern‐Volmer plots indicated a heterogeneous population of tryptophanyl residues with different accessibilities. A competitive inhibitor, imidazolepropionate, quenched fluorescence; the quenching was used to determine the dissociation constant for the enzyme‐inhibitor complex ( K d = 0.20 mM). Kinetic data showed K i = 0.25 mM. Mixed quencher experiments indicated that the tryptophanyl residues quenched by imidazolepropionate were more accessible to acrylamide and less accessible to iodide. These studies suggest that the residues involved in putative energy transfer during photoactivation are not fully exposed.