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Dynamic /sup 13/C NMR investigations of substrate interaction and catalysis by cobalt(II) human carbonic anhydrase I

Technical Report ·
OSTI ID:6068358
;
Using /sup 13/C NMR spectroscopy, the authors further investigated the binding of HCO/sub 3//sup -/ in the active site of an artificial form of human carbonic anhydrase I in which the native zinc is replaced by Co(II). The Co(II) enzyme, unlike all other metal-substituded derivatives, has functional properties closely similar to those of the native zinc enzyme. By measuring the spin-lattice relaxation rate and the line width for both the CO/sub 2/ and HCO/sub 3//sup -/ at two field strengths, both the paramagnetic effects that reflect substrate binding and the exchange effects due to catalysis at chemical equilibrium were determined. The following are the results at 14 C and pH 6.3: HCO/sub 3//sup -/ is bound in the active site of the catalytically competent enzyme with the /sup 13/C of the HCO/sub 3//sup -/ located 3.22 + or - 0.02 A from the Co(II); the apparent equilibrium dissociation constant for the bound HCO/sub 3//sup -/ is 7.6 + or - 1.5 mM, determined by using the paramagnetic effects on the line widths,and 10 + or - 2 mM, determined by using the exchange effects; the lifetime of HCO/sub 3//sup -/ bound to the metal is (4.4 + or - 0.4) x .00001 the overall catalyzed reversible reaction CO/sub 2/ yields HCO/sub 3//sup -/ exchange rate constant of the Co(II) enzyme is (9.6 + or - 0.4) x 1,000/s the electron spin relaxation time of the Co(II), determined by using paramagnetic effects on the bound HCO/sub 3//sup -/, is (1.1 + or - 0.1) x 10/sup -11/ s.
Research Organization:
Naval Medical Research Inst., Bethesda, MD (USA)
OSTI ID:
6068358
Report Number(s):
AD-A-162514/4/XAB; NMRI-85-24
Country of Publication:
United States
Language:
English

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Related Subjects

550200* -- Biochemistry
59 BASIC BIOLOGICAL SCIENCES
BIOCHEMICAL REACTION KINETICS
CARBON 13
CARBON ISOTOPES
CARBON-OXYGEN LYASES
CARBONIC ANHYDRASE
CATALYSIS
COBALT
DISSOCIATION
ELECTRONS
ELEMENTARY PARTICLES
ELEMENTS
ENZYME ACTIVITY
ENZYMES
EVEN-ODD NUCLEI
FERMIONS
FUNCTIONAL ANALYSIS
HYDRO-LYASES
ISOTOPES
KINETICS
LEPTONS
LIGHT NUCLEI
LYASES
MAGNETIC RESONANCE
MAGNETISM
MATHEMATICS
METALS
NMR SPECTRA
NUCLEAR MAGNETIC RESONANCE
NUCLEI
PARAMAGNETISM
REACTION KINETICS
RELAXATION TIME
RESONANCE
SIMULATION
SPECTRA
SPECTROSCOPY
STABLE ISOTOPES
SUBSTRATES
TRANSITION ELEMENTS
ZINC