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/sup 113/Cd NMR of Cd(II)-substituted Zn(II) metalloenzymes

Journal Article · · Fed. Proc.; (United States)
OSTI ID:5724124
;

Cadmium can replace zinc and magnesium in alkaline phosphatase from Escherichia coli, which permits the characterization of the catalytically important metal-binding sites by /sup 113/Cd NMR. At pH 6.5, in the absence of phosphate, two equivalents of cadmium are bound in identical sites (A), one in each monomer. Either raising the pH or phosphorylation of Cd/sub 2/AP (AP is apoalkaline phosphatase) results in migration of Cd(II) from the site A in one monomer to the opposite monomer to occupy a second site (B) adjacent to the A site in the first monomer, a site stabilized by phosphorylation or high pH. At pH 6.5 in the presence of phosphate, the /sup 113/Cd NMR spectrum of Cd/sub 6/AP consists of three narrow resonances from three pairs of fully occupied sites, A, B, and C. The resonances at 153 and 70 ppm represent two metal sites (A and B) 3.9 angstrom apart at each active center and adjacent to the serine phosphorylated during turnover. At this pH the enzyme exists almost exclusively as the covalent phosphoseryl form E-P with a /sup 31/P resonance at approx. 9 ppm. As the pH is raised a /sup 31/P signal from the noncovalent E.P complex appears at approx. 13ppm. This is reflected in the /sup 113/Cd spectrum by a split of both the A- and B-site resonances into pairs, a set at 137 and 65 ppm for E.P, and 153 and 70 ppm for the E-P species. /sup 113/Cd-O-/sup 31/P coupling of 30 Hz on the /sup 31/P resonance of E.P shows the noncovalently bound phosphate to be coordinated to one but not both metal ions at each active site.

Research Organization:
Yale Univ., New Haven, CT
OSTI ID:
5724124
Journal Information:
Fed. Proc.; (United States), Journal Name: Fed. Proc.; (United States) Vol. 41:13; ISSN FEPRA
Country of Publication:
United States
Language:
English

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Related Subjects

550201* -- Biochemistry-- Tracer Techniques
59 BASIC BIOLOGICAL SCIENCES
ALKALINE EARTH METALS
ALKALINE PHOSPHATASE
BACTERIA
BETA DECAY RADIOISOTOPES
BETA-MINUS DECAY RADIOISOTOPES
CADMIUM 113
CADMIUM 115
CADMIUM ISOTOPES
CATIONS
CHARGED PARTICLES
CHEMICAL BONDS
CHEMICAL REACTIONS
DAYS LIVING RADIOISOTOPES
ELEMENTS
ENZYMES
ESCHERICHIA COLI
ESTERASES
EVEN-ODD NUCLEI
HYDROLASES
INTERMEDIATE MASS NUCLEI
IONS
ISOMERIC TRANSITION ISOTOPES
ISOTOPE APPLICATIONS
ISOTOPES
LABELLED COMPOUNDS
LIGHT NUCLEI
MAGNESIUM
MAGNETIC RESONANCE
METALS
MICROORGANISMS
MONOMERS
NMR SPECTRA
NUCLEAR MAGNETIC RESONANCE
NUCLEI
ODD-EVEN NUCLEI
PH VALUE
PHOSPHATASES
PHOSPHORUS 31
PHOSPHORUS ISOTOPES
PHOSPHORYLATION
RADIOISOTOPES
RECEPTORS
RESONANCE
SPECTRA
STABLE ISOTOPES
TRACER TECHNIQUES
YEARS LIVING RADIOISOTOPES
ZINC