Escherichia coli Co(II) Alkaline Phosphatase

Substitution of Co(II) for the native Zn(II) ions of Escherichia coli alkaline phosphatase results in an enzymatically active protein with an intense visible absorption spectrum. The major absorption peaks occur at 640 mµ (e = 260), 605 (e = 220), 555 (e = 378), and 510 (e = 335). Spectrophotometric titration of the apoenzyme with Co(II) reveals two cobalt-binding sites per enzyme dimer which contribute equally to the spectrum. Circular dichroic (CD) spectra show most of these bands to be optically active and reveal additional transitions between 300 and 400 mµ associated with the cobalt chromophores. Phosphate produces a large change in the band structure of the visible absorption spectrum and an inversion of the CD spectrum in the region of 550 mµ. One phosphate molecule per enzyme dimer induces the spectral changes. The visible absorption and CD spectra of both the Co(II) enzyme and its phosphate complex were simultaneously fitted with a series of gaussian absorption bands which reveal that all four spectra may be fitted by 9 bands of the same frequency and band width. Phosphate induces no change in band energies, but changes the relative oscillator strengths and induces large negative ellipticity in a previously optically inactive band. Thus it appears that phosphate produces alterations in the immediate dissymmetrical environment of the Co(II) without major changes in coordination geometry. Computer calculation of the visible optical rotatory dispersion from the resolved CD bands generates curves similar to those observed experimentally. Ultraviolet CD measurements show that neither Co(II) nor phosphate produces major changes in the conformation of the molecule. A mutant alkaline phosphatase, that from E. coli U47, does show major changes in the conformation of the metal-binding site and significant changes in the conformation of the peptide backbone.