title: Fluorescence properties of hog kidney aminoacylase I.
abstract: The state of the tryptophan residues of porcine kidney aminoacylase I (EC 3.5.1.14) was investigated by fluorescence spectroscopy and chemical modification. The pH-dependence of the fluorescence emission spectrum of the enzyme indicates that its native conformation prevails between pH 6 and 9.5. Within this range, the ionization of a residue with an apparent pKa of 7.1 quenches the enzyme fluorescence by about 15%. A similar reduction of fluorescence intensity accompanies the inactivation of aminoacylase I by treatment with N-bromosuccinimide in low excess. This suggests that in both cases a single tryptophyl residue out of eight residues per subunit is affected. Quenching by iodide revealed that, in the native conformation of the enzyme, 5-6 tryptophans per subunit are accessible, while 2-3 are buried within the protein. 8-Anilinonaphthalene-1-sulfonate (ANS) is tightly bound to aminoacylase I (1 mol/mol dimer, Kd less than 1 microM). ANS binding does not interfere with substrate turnover; the spectroscopic properties of the aminoacylase-ANS complex are consistent with bound ANS being excited by radiationless energy transfer (RET) from buried tryptophyl residues of the enzyme.
authors.affiliation: Institut für Physiologische Chemie, Philipps-Universität, Marburg, (Lahn), Bundesrepublik Deutschland.
authors.forename: T
authors.initials: T
authors.lastname: vom Bruch
authors.forename: K H
authors.initials: KH
authors.lastname: Röhm
mesh_terms: D000581:Amidohydrolases / Q000378:metabolism*; D000817:Anilino Naphthalenesulfonates; D000818:Animals; D005456:Fluorescent Dyes; D006863:Hydrogen-Ion Concentration; D007668:Kidney / Q000201:enzymology*; D007700:Kinetics; D008024:Ligands; D013050:Spectrometry, Fluorescence / Q000379:methods; D013552:Swine; D014364:Tryptophan / Q000032:analysis
chemical_list: D000817:Anilino Naphthalenesulfonates; D005456:Fluorescent Dyes; D008024:Ligands; C027132:1-anilino-8-naphthalenesulfonate; D014364:Tryptophan; D000581:Amidohydrolases; C019672:aminoacylase I