GA3- PREPARATION AND CHARACTERIZATION OF THE GA3+FE2+ AND GA3+ZN2+ FORMS OF BOVINE SPLEEN PURPLE ACID-PHOSPHATASE( AS A FUNCTIONAL SUBSTITUTE FOR FE3+ )

A general method has been developed that allows the specific substitut ion of both iron atoms in the enzyme bovine spleen purple acid phospha tase (BSPAP), which possesses a dinuclear iron center at the active si te. The approach is demonstrated by the preparation and characterizati on (atomic absorption spectrometry, enzyme kinetics, optical spectrosc opy, and electron paramagnetic resonance spectroscopy) of two metal-su bstituted forms in which the ferric iron has been replaced by Ga3+: Ga 3+Fe2+-BSPAP and Ga3+Zn2+-BSPAP. Both forms are colorless but exhibit enzymatic activity comparable to that of the native Fe3+Fe2+-BSPAP. Sm all but consistent changes in kinetics parameters and pH profiles were detected both upon substitution of Fe3+ by Ga3+ and upon substitution of Fe2+ by Zn2+. These results constitute the first evidence that the diamagnetic Ga3+ ion can serve as a functional analogue of Fe3+ in an enzyme, and suggest a novel approach for the study of the role of Fe3 + in other iron enzymes.