Characterization of the Properties of the Multiple Metal Binding Sites in Alkaline Phosphatase by Carbon-13 Nuclear Magnetic Resonance

James D. Otvos, Ian M. Armitage

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Carbon-13 nuclear magnetic resonance (13C NMR) of Escherichia coli alkaline phosphatase labeled biosynthetically with β, β-[γ-13C] dideuteriohistidine has been used to determine the number and identity of the histidine residues that participate in metal ion coordination at the three classes of binding sites in this dimeric Zn2+metalloenzyme. Detailed13C NMR titrations of the apoenzyme with113Cd2+and Mg2+, in conjunction with parallel113Cd NMR measurements [Otvos, J. D., & Armitage, I. M. (1980) Biochemistry (third of three papers in this issue)], permitted the assignment of four histidine residues as ligands to the “catalytic”, or A site, metal ions, two coordinated via their NTimidazole nitrogens and two via NT. in addition, a fifth histidyl ligand, coordinated through NT, was shown to be located at the “structural”, or B, sites on the dimer. The “regulatory”, or C, sites do not contain histidyl metal ligands Unambiguous identification of the three histidines coordinated to metal ion via NTwas provided by the observation of resolved113Cd-13C spin-spin coupling (3J = 12-19 Hz) in their γ-carbon resonances. Once assigned, the13C resonances of the five histidyl metal ligands were used to monitor the relative affinities of the A, B, and C sites for Cd2+and Zn2+. At pH 6.3, Cd2+was found to bind to the A sites at least 10 times tighter than to the B or C sites, which have roughly equal affinities. in marked contrast, Zn2+was found to have similar affinities for the A and B sites at both pH 6.3 and 8.0. The affinity of the C sites for Zn2+and Mg2+was shown to be at least an order of magnitude lower. The binding constants of all three sites for Cd2+and Zn2+are greater than 105M−1. Evidence is also presented that suggests that the A, B, and C sites may be located in close proximity to one another in the monomers.

Original languageEnglish (US)
Pages (from-to)4021-4030
Number of pages10
Issue number17
StatePublished - Aug 1 1980
Externally publishedYes


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