J. Biol. Chem., Vol. 259, Issue 8, 4987-4990, Apr, 1984
113Cd NMR. Arsenate binding to Cd(II) alkaline phosphatase
P Gettins and JE Coleman
Alkaline phosphatase from Escherichia coli contains three metal binding
sites (A, B, and C) located at sites forming a triangle with sides of 4, 5,
and 7 A (Wyckoff, H.W., Handschumacher, M., Murthy, K., and Sowadski, J.M.
(1983) Adv. Enzymol. 55, 453). When all three sites are occupied by Cd(II)
the enzyme has a very low turnover; at least 10(3) slower than the native
Zn(II) enzyme. The slow turnover number has made the Cd(II) enzyme useful
in NMR studies of the mechanism of alkaline phosphatase. The binding of
arsenate to two forms of Cd(II) alkaline phosphatase (Cd(II)2alkaline
phosphatase and Cd(II)6alkaline phosphatase) has been studied by 113Cd NMR.
Cd(II)2alkaline phosphatase, pH 6.3, binds arsenate at only one monomer of
the dimeric enzyme and causes migration of Cd(II) from the A site of one
monomer to the B site of the arsenylated monomer. This same migration has
previously been observed to accompany metal ion-dependent phosphate
binding, but is much more rapid in the case of arsenate. The acceleration
of migration induced by arsenate supports the conclusion based on the
phosphate data that the substrate anion binds to the A site metal ion of
one monomer prior to migration and that only the metal ion at A site is
required for phosphorylation (arsenylation) of serine 102. The 113Cd
chemical shifts of A and B site metal ions are very sensitive to the form
of the bound arsenate, i.e. covalent (E-As) or noncovalent (E X As)
complex. Like the analogous phosphate derivatives, the change of chemical
shift of A site (to which phosphate is coordinated in the E X P complex) is
much greater than that of the B site metal ion, when the arsenate shifts
between the two intermediates, suggesting that arsenate is also coordinated
to A site in the E X As intermediate. The chemical shifts of A and B site
113Cd(II) ions are considerably different in the arsenate and phosphate
derivatives, while the C site 113Cd(II) ions have nearly identical chemical
shifts. Thus the substrate appears to interact closely with both A and B
sites, while C site appears relatively unimportant in phosphomonoester
hydrolysis. The analogous behavior of arsenate and phosphate at the active
center as evaluated by 113Cd NMR supports the validity of using the heavier
arsenate derivative in x-ray diffraction studies.
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