title: Tetracyanonickelate probes the active site of sulfur-free rhodanese.
abstract: Tetracyanonickelate (Ni(CN)4(2-)) was used as a probe for the active site of sulfur-free rhodanese (E) in physical and kinetic studies. Ni(CN)4(2-) quenches the intrinsic fluorescence as well as the fluorescence of enzyme-bound 2-anilinonaphthalene-8-sulfonic acid (2,8-ANS), an inhibitor that is competitive with respect to thiosulfate. A facile binding method based on centrifugation was developed to study Ni(CN)4(2-) binding to E. Binding studies performed using either of the electrophoretic variants A and B, fractionated by DE52 column chromatography, showed one high affinity Ni(CN)4(2-)-binding site in each species and additional weak sites on the more electropositive form A. The high affinity Ni(CN)4(2-) binding was corroborated by ultrafiltration binding (Kd = 3.95 +/- 0.35 microM), titration of intrinsic fluorescence (Kd = 1.8 +/- 0.11 microM), and displacement of enzyme-bound 2,8-ANS (Kd = 1.9 +/- 1.1 microM). A nonlinear least squares analysis of kinetic data collected under conditions used for the binding studies gave a Ni(CN)4(2-) inhibition constant of 21 microM. It is concluded that Ni(CN)4(2-) binds to sulfur-free rhodanese in solution near the active site as has been shown in x-ray crystal studies (Lijk, L. J., Kalk, K. H., Brandenburger, N. P., and Hol, W. G. J. (1983) Biochemistry 22, 2952-2957). In keeping with recent suggestions that the conformational state of the enzyme is dynamically determined, the discrepancy between Ni(CN)4(2-) affinity as determined by physical methods and that by kinetic methods suggests that Ni(CN)4(2-) may be able to distinguish the conformation of the working enzyme from those of the idle forms.
mesh_terms: D001665:Binding Sites; D003460:Crystallization; D003486:Cyanides / Q000378:metabolism*; D007700:Kinetics; D008956:Models, Chemical; D009532:Nickel / Q000378:metabolism*; D011190:Potassium Cyanide / Q000494:pharmacology; D013050:Spectrometry, Fluorescence; D013455:Sulfur / Q000378:metabolism*; D013466:Sulfurtransferases / Q000378:metabolism*; D013884:Thiosulfate Sulfurtransferase / Q000378:metabolism*
chemical_list: D003486:Cyanides; C047503:tetracyanonickelate; D013455:Sulfur; D009532:Nickel; D013466:Sulfurtransferases; D013884:Thiosulfate Sulfurtransferase; D011190:Potassium Cyanide