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.