			Home About us Contact

Human Carbonic Anhydrase II (human + carbonic_anhydrase_ii)

Distribution by Scientific Domains

Chemistry	100%

Selected Abstracts

Production and X-ray crystallographic analysis of fully deuterated human carbonic anhydrase II

ACTA CRYSTALLOGRAPHICA SECTION F (ELECTRONIC), Issue 1 2006
Monika Budayova-Spano
Human carbonic anhydrase II (HCA II) is a zinc metalloenzyme that catalyzes the reversible hydration and dehydration of carbon dioxide and bicarbonate, respectively. The rate-limiting step in catalysis is the intramolecular transfer of a proton between the zinc-bound solvent (H2O/OH,) and the proton-shuttling residue His64. This distance (,7.5,Å) is spanned by a well defined active-site solvent network stabilized by amino-acid side chains (Tyr7, Asn62, Asn67, Thr199 and Thr200). Despite the availability of high-resolution (,1.0,Å) X-ray crystal structures of HCA II, there is currently no definitive information available on the positions and orientations of the H atoms of the solvent network or active-site amino acids and their ionization states. In preparation for neutron diffraction studies to elucidate this hydrogen-bonding network, perdeuterated HCA II has been expressed, purified, crystallized and its X-ray structure determined to 1.5,Å resolution. The refined structure is highly isomorphous with hydrogenated HCA II, especially with regard to the active-site architecture and solvent network. This work demonstrates the suitability of these crystals for neutron macromolecular crystallography. [source]

Characterization of carbonic anhydrase from Neisseria gonorrhoeae

FEBS JOURNAL, Issue 6 2001
Björn Elleby
We have investigated the steady state and equilibrium kinetic properties of carbonic anhydrase from Neisseria gonorrhoeae (NGCA). Qualitatively, the enzyme shows the same kinetic behaviour as the well studied human carbonic anhydrase II (HCA II). This is reflected in the similar pH dependencies of the kinetic parameters for CO2 hydration and the similar behaviour of the kinetics of 18O exchange between CO2 and water at chemical equilibrium. The pH profile of the turnover number, kcat, can be described as a titration curve with an exceptionally high maximal value of 1.7 × 106 s,1 at alkaline pH and a pKa of 7.2. At pH 9, kcat is buffer dependent in a saturable manner, suggesting a ping-pong mechanism with buffer as the second substrate. The ratio kcat/Km is dependent on two ionizations with pKa values of 6.4 and 8.2. However, an 18O-exchange assay identified only one ionizable group in the pH profile of kcat/Km with an apparent pKa of 6.5. The results of a kinetic analysis of a His66,Ala variant of the bacterial enzyme suggest that His66 in NGCA has the same function as a proton shuttle as His64 in HCA II. The kinetic defect in the mutant can partially be overcome by certain buffers, such as imidazole and 1,2-dimethylimidazole. The bacterial enzyme shows similar Ki values for the inhibitors NCO,, SCN, and N3, as HCA II, while CN, and the sulfonamide ethoxzolamide are considerably weaker inhibitors of the bacterial enzyme than of HCA II. The absorption spectra of the adducts of Co(II)-substituted NGCA with acetazolamide, NCO,, SCN,, CN, and N3, resemble the corresponding spectra obtained with human Co(II)-isozymes I and II. Measurements of guanidine hydrochloride (GdnHCl)-induced denaturation reveal a sensitivity of the CO2 hydration activity to the reducing agent tris(2-carboxyethyl)phosphine (TCEP). However, the A292/A260 ratio was not affected by the presence of TCEP, and a structural transition at 2.8,2.9 m GdnHCl was observed. [source]

Emerging from pseudo-symmetry: the redetermination of human carbonic anhydrase II in monoclinic P21 with a doubled a axis

ACTA CRYSTALLOGRAPHICA SECTION D, Issue 8 2010
Arthur H. Robbins
The crystal structure of human carbonic anhydrase II in the monoclinic P21 space group with a doubled a axis from that of the usually observed unit cell has recently been reported, with one of the two molecules in the asymmetric unit demonstrating rotational disorder [Robbins et al. (2010), Acta Cryst. D66, 628,634]. The structure has been redetermined, with the coordinates of both pseudo-symmetrically related molecules in the crystallographic asymmetric unit translated by x, = x± 1/4, and no rotational disorder is observed. This corresponds to a different choice of how the four molecules in the unit cell should be grouped into pairs that represent a single asymmetric unit. [source]

Structure of a monoclinic polymorph of human carbonic anhydrase II with a doubled a axis

ACTA CRYSTALLOGRAPHICA SECTION D, Issue 5 2010
Arthur H. Robbins
The crystal structure of human carbonic anhydrase II with a doubled a axis from that of the usually observed monoclinic unit cell has been determined and refined to 1.4,Å resolution. The diffraction data with h = 2n + 1 were systematically weaker than those with h = 2n. Consequently, the scaling of the data, structure solution and refinement were challenging. The two molecules comprising the asymmetric unit are related by a noncrystallographic translation of ½ along a, but one of the molecules has two alternate positions related by a rotation of approximately 2°. This rotation axis is located near the edge of the central ,-sheet, causing a maximum distance disparity of 1.7,Å between equivalent atoms on the diametrically opposite side of the molecule. The crystal-packing contacts are similar to two sequential combined unit cells along a of the previously determined monoclinic unit cell. Abnormally high final Rcryst and Rfree values (20.2% and 23.7%, respectively) are not unusual for structures containing pseudo-translational symmetry and probably result from poor signal to noise in the weak h -odd data. [source]

High-resolution structure of human carbonic anhydrase II complexed with acetazolamide reveals insights into inhibitor drug design

ACTA CRYSTALLOGRAPHICA SECTION F (ELECTRONIC), Issue 10 2009
Katherine H. Sippel
The crystal structure of human carbonic anhydrase II (CA II) complexed with the inhibitor acetazolamide (AZM) has been determined at 1.1,Å resolution and refined to an Rcryst of 11.2% and an Rfree of 14.7%. As observed in previous CA II,inhibitor complexes, AZM binds directly to the zinc and makes several key interactions with active-site residues. The high-resolution data also showed a glycerol molecule adjacent to the AZM in the active site and two additional AZMs that are adventitiously bound on the surface of the enzyme. The co-binding of AZM and glycerol in the active site demonstrate that given an appropriate ring orientation and substituents, an isozyme-specific CA inhibitor may be developed. [source]

Preliminary joint neutron and X-ray crystallographic study of human carbonic anhydrase II

ACTA CRYSTALLOGRAPHICA SECTION F (ELECTRONIC), Issue 5 2009
S. Z. Fisher
Carbonic anhydrases catalyze the interconversion of CO2 to HCO3,, with a subsequent proton-transfer (PT) step. PT proceeds via a proposed hydrogen-bonded water network in the active-site cavity that is stabilized by several hydrophilic residues. A joint X-ray and neutron crystallographic study has been initiated to determine the specific water network and the protonation states of the hydrophilic residues that coordinate it in human carbonic anhydrase II. Time-of-flight neutron crystallographic data have been collected from a large (,1.2,mm3) hydrogen/deuterium-exchanged crystal to 2.4,Å resolution and X-ray crystallographic data have been collected from a similar but smaller crystal to 1.5,Å resolution. Obtaining good-quality neutron data will contribute to the understanding of the catalytic mechanisms that utilize water networks for PT in protein environments. [source]