The β-carbonic anhydrase (HICA) allosteric site variants V47A and G41A were overexpressed and purified to homogeneity. X-ray crystallographic constructions of Type I (non-allosteric) β-CAs and the D44N variant of HICA suggest that Type II β-CAs can also adopt an active R-state conformation in which Asp44 pairs with Arg46 permitting the coordination of the catalytically essential water molecule to the active site zinc ion (3). The noncatalytic bicarbonate ion is definitely believed to stabilize the inactive T state (Plan 1) and this hypothesis is definitely borne out from the W39F variant of HICA in which bicarbonate ion is definitely significantly less effective in inhibiting the enzyme (3). Plan 1 Structural schematic of important active site and AZD1152-HQPA noncatalytic bicarbonate binding site relationships in the hypothesized active (R-state) and inactive (T-state) conformations of HICA (2). A key player in AZD1152-HQPA the allosteric (T → R) transition of HICA in Plan I is definitely Val47: the steric bulk of this side chain is definitely hypothesized to displace bicarbonate from your allosteric binding site in the R state and thus provides the necessary steric coupling mechanism between the bicarbonate binding and the used allosteric state. In addition examination of X-ray crystallographic constructions of type I and type II β-CA discloses that type I β-CAs all have a slightly more heavy Ala41 residue rather than the smaller Gly41 in type II β-CAs. Examination of type II β-CA constructions with Gly41 modified to Ala suggests that this structural switch may preclude bicarbonate binding AZD1152-HQPA to the allosteric site (1). To investigate the functions of these residues we kinetically and structurally characterized the G41A and V47A variants of HICA. Surprisingly we find that these variants have little impact on the catalytic function of HICA. However we have serendipitously discovered that these variants are able to bind bicarbonate ion in an intermediate binding site that apparently defines the access/exit pathway of bicarbonate to/from the allosteric site. These results suggest that the mechanism of allosteric inhibition of HICA and the selectivity of the allosteric binding site is definitely Tmem10 more complex than previously acknowledged. Experimental Procedures Manifestation and purification of wild-type and recombinant enzymes Wild type HICA was prepared as previously explained (2). Site-directed mutations of the gene coding for HICA were constructed using megaprimer PCR (4) with (New England Biolabs) or turbo (Stratagene) polymerase and commercial oligonucleotides (Integrated DNA Techonolgies). For variant V47A a mutated oligonucleotide2 5′-TTCAGCAGGCCGCACGGCTATC-3′ was combined with the 5′ oligonucleotide primer PHI1X (5′-TGCCCATGGATAAAATTAAACAACTCTTT-3) in the 1st PCR reaction to give a 129 bp product. This PCR product was used like a megaprimer in a second PCR reaction with the 3′ oligonucleotide primer PHI2X (5′-TGCCTGCAGTTATTATGTATTTTCAAGATG-3′) to produce the final mutated HICA gene. The final PCR product was digested with and were determined by non-linear least squares suits to [CO2] data using Source 7.0 (Microcal). For those kinetics measurements reported here substrate dependence of CO2 hydration rates appeared to follow Michaelis-Menten kinetics. The kinetic constants and are reported here on a per subunit basis. Crystallographic methods Starlike clusters of orthorhombic plates of HICA-V47A appeared after 3 months in 1.7 M ammonium sulfate 4 PEG 400 0.1 M HEPES pH 7.50 6 mg/mL protein at 4 °C using hanging drop vapor diffusion. Crystals were soaked in artificial mother liquor plus 30% glucose for 30-60 mere seconds prior to adobe flash chilling in liquid nitrogen. Data collection for PDB 3E2X was carried out at beamline F2 of CHESS at a wavelength of 0.98 ? detector using 0.5° oscillations at a temperature of 100 K. Tetragonal crystals of HICA-V47A were cultivated in 2-3 days in 0.7 M sodium potassium tartrate 0.1 M HEPES pH 7.50 12 mg/mL protein at 22 °C. Crystals were soaked AZD1152-HQPA for 1-2 moments in artificial mother liquor plus either 30% glucose (PDB 3E31) or 30% glucose and 100 mM NaHCO3 (PDB 3E3F) before adobe flash chilling in liquid nitrogen. Data collection was carried out at beamline F2 of CHESS at a wavelength of 0.98 ? detector using 0.5° oscillations at a temperature of 100 K. Monoclinic crystals of HICA-G41A were cultivated in 2-3 days in 1.8 M ammonium sulfate 4 PEG-400 0.1 M HEPES pH 7.50 12 mg/mL protein at 22 °C. Crystals were soaked for 1-2 moments.