The enzyme catalyses the reversible hydration of gaseous CO2 to carbonic acid, which dissociates to give hydrogencarbonate above neutral pH. It is widespread and found in archaea, bacteria, and eukaryotes. Three distinct classes exist, and appear to have evolved independently. Contains zinc.
The enzyme catalyses the reversible hydration of gaseous CO2 to carbonic acid, which dissociates to give hydrogencarbonate above neutral pH. It is widespread and found in archaea, bacteria, and eukaryotes. Three distinct classes exist, and appear to have evolved independently. Contains zinc.
type II beta-carbonic anhydrases have a characteristic triad of non-catalytic residues, i.e. Trp39, Arg64, and Tyr181 in HICA, about 8 A away from the active site zinc ion that can bind bicarbonate ion. The studied anion binding site along the dimerization interface of HICA is an escort site that represents an intermediate along the ingress/egress route of bicarbonate ion to/from the allosteric binding site, overview. The mechanism of sulfate activation of HICA is the result of sulfate ion competing for bicarbonate at this escort site, preventing passage of bicarbonate from bulk solution to its allosteric site. The noncatalytic bicarbonate ion is a negative allosteric effector that stabilizes an inactive, T-state conformation of the enzyme, where Asp44 displaces the catalytically essential zinc-bound water. Type II beta-CAs can also adopt an active, R-state conformation in which Asp44 pairs with Arg46, allowing the coordination of the catalytically essential water molecule to the active site zinc ion. The noncatalytic bicarbonate ion stabilizes the inactive T state. The steric bulk of the Val47 side chain displaces bicarbonate from the allosteric binding site in the R state, and thus provides the necessary steric coupling mechanism between the bicarbonate binding and the adopted allosteric state
type II beta-carbonic anhydrases have a characteristic triad of non-catalytic residues, i.e. Trp39, Arg64, and Tyr181 in HICA, about 8 A away from the active site zinc ion that can bind bicarbonate ion. The studied anion binding site along the dimerization interface of HICA is an escort site that represents an intermediate along the ingress/egress route of bicarbonate ion to/from the allosteric binding site, overview. The mechanism of sulfate activation of HICA is the result of sulfate ion competing for bicarbonate at this escort site, preventing passage of bicarbonate from bulk solution to its allosteric site. The noncatalytic bicarbonate ion is a negative allosteric effector that stabilizes an inactive, T-state conformation of the enzyme, where Asp44 displaces the catalytically essential zinc-bound water. Type II beta-CAs can also adopt an active, R-state conformation in which Asp44 pairs with Arg46, allowing the coordination of the catalytically essential water molecule to the active site zinc ion. The noncatalytic bicarbonate ion stabilizes the inactive T state. The steric bulk of the Val47 side chain displaces bicarbonate from the allosteric binding site in the R state, and thus provides the necessary steric coupling mechanism between the bicarbonate binding and the adopted allosteric state
the sulfate ion competes with the bicarbonate ion for the binding site near the active site affecting the allosteric regulation of the enzyme, binding structure in wild-type an dmutant enzymes, overview
preparation of a Co(II)-substituted HICA, Co-HICA. Co(II)-substituted HICA, Co-HICA, has comparable, 20% enhanced kcat and 2.3fold increased Km/kcat compared to that of the wild-type enzyme
pH profile, pH-dependent changes in the absorption spectrum of Co-HICA including an increase in molar absorptivity and a red shift of a 580 nm peak with decreasing pH, correlate with the pH-dependence of kcat/Km, overview
recombinant Co-HICA, the crystal structure shows a four-coordinate geometry for Co-HICA with pH-dependent changes in the absorption spectrum of Co-HICA, overview
the studied anion binding site along the dimerization interface of HICA is an escort site that represents an intermediate along the ingress/egress route of bicarbonate ion to/from the allosteric binding site, overview. Type II beta-CAs can also adopt an active, R-state conformation in which Asp44 pairs with Arg46, allowing the coordination of the catalytically essential water molecule to the active site zinc ion. The noncatalytic bicarbonate ion stabilizes the inactive T state
the studied anion binding site along the dimerization interface of HICA is an escort site that represents an intermediate along the ingress/egress route of bicarbonate ion to/from the allosteric binding site, overview. Type II beta-CAs can also adopt an active, R-state conformation in which Asp44 pairs with Arg46, allowing the coordination of the catalytically essential water molecule to the active site zinc ion. The noncatalytic bicarbonate ion stabilizes the inactive T state
overall secondary and tertiary structure of Co-HICA differ from wild-type Zn-enzyme structure, active R-state and inactive T-state conformations, overview
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CRYSTALLIZATION (Commentary)
ORGANISM
UNIPROT
LITERATURE
purified wild-type and mutant enzymes, 12 mg/ml protein mixed with 0.7 M sodium potassium tartrate, 0.10 M HEPES, pH 7.5, for tetragonal crystals and with 1.8 M ammonium sulfate, 4% PEG 400, 0.10 M HEPES, pH 7.5, for monoclinic crystals, 22°C, 2-3 days, crystals are soaked for 1-2 min in artificial mother liquor plus either 30% glucose or 30% glucose and 100 mM NaHCO3, X-ray diffraction structure determination and analysis
purified recombinant Co-HICA by hanging drop vapor diffusion, 10 mg/ml protein crystallized in 0.2 M sodium acetate, 0.1 M Tris-HCl, pH 8.5, 0.1 M (NH4)2SO4, and 27% PEG 4000, 22°C, several days, X-ray diffraction structure determination and analysis at 2.5 A resolution
the mutant has kcat/Km values similar to wild-type enzyme, and exhibits a similar dramatic decrease in catalytic activity at pH values below pH 8.0, but HICA-G41A is serendipitously found to bind sulfate ion or bicarbonate ion near pairs of Glu50 and Arg64 residues located on the dimerization interface, 2 of 12 chains in the asymmetric unit bind bicarbonate ion exclusively at the dimerization interface, while the remaining 10 chains bind bicarbonate ion exclusively at the allosteric site
the mutant has kcat/Km values similar to wild-type enzyme, and exhibits a similar dramatic decrease in catalytic activity at pH values below pH 8.0, but HICA-V47A is serendipitously found to bind sulfate ion or bicarbonate ion near pairs of Glu50 and Arg64 residues located on the dimerization interface, bicarbonate ions simultaneously bind to both the dimerization interface and the allosteric sites