A spectroscopic investigation of cobalt(II) substituted alkaline phosphatase.
The electronic and 1H NMR spectra are reported for the cobalt(II) alkaline phosphatase (EC 3.1.3.1.) system at pH around 6 in the range 0-2 mol of cobalt per mol protein. It is shown that under the present experimental conditions cobalt(II) selectively populates the A sites. Three isotropically shifted NH signals have been detected in the A site that indicate the presence of three histidines in the coordination sphere of cobalt(II). The electronic spectra and the nuclear relaxation properties are consistent with pentacoordination of cobalt(II) in the A site. The finding of reproducible preparation routes for the derivatives, and of appropriate experimental conditions for the observation of their 1H NMR spectra, open new possibilities for the spectroscopic investigation of alkaline phosphatase.
Banci
L
L
Bertini
I
I
Gallori
E
E
Luchinat
C
C
Paoletti
F
F
Polsinelli
M
M
Viezzoli
M S
MS
D000469:Alkaline Phosphatase / Q000378:metabolism*; D003035:Cobalt / Q000378:metabolism*; D004926:Escherichia coli / Q000201:enzymology; D009682:Magnetic Resonance Spectroscopy; D011485:Protein Binding; D013053:Spectrophotometry
D003035:Cobalt; D000469:Alkaline Phosphatase