title: Dynamic 13C NMR investigations of substrate interaction and catalysis by cobalt(II) human carbonic anhydrase I.
abstract: Using 13C NMR spectroscopy, we have further investigated the binding of HCO3- 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-substituted 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 CO2 and HCO3- at two field strengths, we have determined both the paramagnetic effects that reflect substrate binding and the exchange effects due to catalysis at chemical equilibrium. The following are the results at 14 degrees C and pH 6.3 (1) HCO3- is bound in the active site of the catalytically competent enzyme with the 13C of the HCO3- located 3.22 +/- 0.02 A from the Co(II); (2) the apparent equilibrium dissociation constant for the bound HCO3- is 7.6 +/- 1.5 mM, determined by using the paramagnetic effects on the line widths, and 10 +/- 2 mM, determined by using the exchange effects; (3) the lifetime of HCO3- bound to the metal is (4.4 +/- 0.4) X 10(-5) s; (4) the overall catalyzed CO2 in equilibrium HCO3- exchange rate constant of the Co(II) enzyme is (9.6 +/- 0.4) X 10(3) s-1; (5) the electron spin relaxation time of the Co(II), determined by using paramagnetic effects on the bound HCO3-, is (1.1 +/- 0.1) X 10(-11) s. The data did not provide any direct information on the binding of CO2.(ABSTRACT TRUNCATED AT 250 WORDS)
authors.forename: T J
authors.initials: TJ
authors.lastname: Williams
authors.forename: R W
authors.initials: RW
authors.lastname: Henkens
mesh_terms: D001639:Bicarbonates / Q000378:metabolism; D001665:Binding Sites; D002247:Carbon Isotopes; D002256:Carbonic Anhydrases / Q000378:metabolism*; D003035:Cobalt / Q000378:metabolism*; D007700:Kinetics; D009682:Magnetic Resonance Spectroscopy / Q000379:methods; D011485:Protein Binding; D013816:Thermodynamics
chemical_list: D001639:Bicarbonates; D002247:Carbon Isotopes; D003035:Cobalt; D002256:Carbonic Anhydrases