Spectroscopic and kinetics studies of a high-salt-stabilized form of the purple acid phosphatase from bovine spleen.

Use of a revised purification procedure that maintains the enzyme in a high-salt environment has resulted in the isolation of a new form of the bovine spleen purple acid phosphatase. This enzyme cannot be distinguished from that previously described [Davis, J. C., Lin, S. S., & Averill, B. A. (1981) Biochemistry 20, 4062] by electrophoresis, isoelectric focusing, Western blot analysis, or N-terminal amino acid sequence and exhibits identical catalytic properties and EPR spectra in the reduced (pink) form. It does, however, possess a much more highly ordered structure as shown by CD spectra and exhibits markedly different reactivity upon oxidation and different visible spectra upon binding of inhibitory anions or changing pH. The properties of the new high-salt-stabilized form of the enzyme have permitted an extensive examination of the visible absorption spectra of complexes of the oxidized and reduced enzyme with inhibitory anions. It is found that these anions may be grouped into three classes on the basis of their effect on the visible absorption maximum and their sensitivity to pH: phosphate, arsenate, and AMP; tungstate and molybdate; and fluoride. This grouping is reinforced by a detailed examination of the steady-state kinetics of the enzyme in the presence of these inhibitors, which reveals that the first class exhibits mixed-type inhibition due to the presence of competitive and noncompetitive binding sites, while the second class exhibits simple non-competitive inhibition. Fluoride exhibits complex inhibition behavior characterized by curved Lineweaver-Burk plots; this behavior cannot be attributed to the presence of inhibitory aluminum fluoride complexes. Taken together, the spectral and kinetics data are consistent with a picture in which tetrahedral oxyanions bind in a noncompetitive fashion by bridging the two iron atoms in the dinuclear center, with the smaller anions also being able to bind in a competitive manner at a single iron atom.