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Literature summary for 4.2.1.1 extracted from

  • Rowlett, R.S.; Hoffmann, K.M.; Failing, H.; Mysliwiec, M.M.; Samardzic, D.
    Evidence for a bicarbonate escort site in Haemophilus influenzae beta-carbonic anhydrase (2010), Biochemistry, 49, 3640-3647.
    View publication on PubMedView publication on EuropePMC

Cloned(Commentary)

Cloned (Comment) Organism
overexpression of wild-type enzyme and mutants V47A and G41A in Escherichia coli Haemophilus influenzae

Crystallization (Commentary)

Crystallization (Comment) Organism
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 Haemophilus influenzae

Protein Variants

Protein Variants Comment Organism
G41A 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 Haemophilus influenzae
V47A 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 Haemophilus influenzae

KM Value [mM]

KM Value [mM] KM Value Maximum [mM] Substrate Comment Organism Structure
additional information
-
additional information steady-state kinetics, overview Haemophilus influenzae

Metals/Ions

Metals/Ions Comment Organism Structure
sulfate 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 Haemophilus influenzae
Zn2+ a zinc-metalloenzyme Haemophilus influenzae

Natural Substrates/ Products (Substrates)

Natural Substrates Organism Comment (Nat. Sub.) Natural Products Comment (Nat. Pro.) Rev. Reac.
H2CO3 Haemophilus influenzae
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CO2 + H2O
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r

Organism

Organism UniProt Comment Textmining
Haemophilus influenzae P45148
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-

Purification (Commentary)

Purification (Comment) Organism
recombinant mutants V47A and G41A to homogeneity from Escherichia coli by ion exchange and hydrophobic interaction chromatography, and gel filtration Haemophilus influenzae

Reaction

Reaction Comment Organism Reaction ID
H2CO3 = CO2 + H2O reaction mechanism, overview Haemophilus influenzae

Substrates and Products (Substrate)

Substrates Comment Substrates Organism Products Comment (Products) Rev. Reac.
H2CO3
-
Haemophilus influenzae CO2 + H2O
-
r
H2CO3 bicarbonate binding mode and binding structure, overview Haemophilus influenzae CO2 + H2O
-
r
additional information 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 Haemophilus influenzae ?
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?

Subunits

Subunits Comment Organism
More 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 Haemophilus influenzae

Synonyms

Synonyms Comment Organism
beta-carbonic anhydrase
-
Haemophilus influenzae
HICA
-
Haemophilus influenzae
More HICA is a type II member of the beta-carbonic anhydrase family Haemophilus influenzae

Temperature Optimum [°C]

Temperature Optimum [°C] Temperature Optimum Maximum [°C] Comment Organism
25
-
assay at Haemophilus influenzae

General Information

General Information Comment Organism
evolution HICA is a type II member of the beta-carbonic anhydrase family Haemophilus influenzae