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HCA II (hca + ii)
Selected AbstractsCharacterization of carbonic anhydrase from Neisseria gonorrhoeaeFEBS JOURNAL, Issue 6 2001Björn 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] Structure of human salivary ,-amylase crystallized in a C -centered monoclinic space groupACTA CRYSTALLOGRAPHICA SECTION F (ELECTRONIC), Issue 2 2006S. Zoë Fisher Human salivary ,-amylase (HSA) is a major secretory protein component of saliva and has important biological functions, including the initial digestion of starch. HSA acts as a monomer and mediates the hydrolysis of ,-1,4-glucosidic linkages in oligosaccharides. To date, all published crystal structures of HSA have been crystallized as monomers in space group P212121. Here, the serendipitous purification, crystallization and ultimate structure determination of a HSA non-crystallographic symmetry (NCS) dimer, while attempting to purify human carbonic anhydrase VI (HCA VI) from saliva using an affinity resin for ,-class carbonic anhydrases, is presented. On further investigation, it was shown that HSA could only be copurified using the affinity resin in the presence of HCA VI which is glycosylated and not the non-glycosylated HCA II. The identification of the HSA crystals was carried out by peptide mapping and mass spectrometry. HSA was shown to have crystallized as an NCS dimer in space group C2, with unit-cell parameters a = 150.9, b = 72.3, c = 91.3,Å, , = 102.8°. The NCS dimer crystal structure is reported to 3.0,Å resolution, with a refined Rcryst of 0.228. The structure is compared with the previously reported P212121 monomer structures and the crystal packing and dimer interface are discussed. [source] Production and X-ray crystallographic analysis of fully deuterated human carbonic anhydrase IIACTA CRYSTALLOGRAPHICA SECTION F (ELECTRONIC), Issue 1 2006Monika 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] Carbonic Anhydrase Inhibitors: Inhibition of Human Erythrocyte Isozymes I and II with a Series of Phenolic AcidsCHEMICAL BIOLOGY & DRUG DESIGN, Issue 5 2010S. Beyza Öztürk Sar, kaya The inhibitory effects of some phenolic acids on the cytosolic human carbonic anhydrase (hCA, EC 4.2.1.1) isozymes hCA I and hCA II were investigated. Ellagic acid, gallic acid, ferulic acid, caffeic acid, quercetin, p -coumaric acid, p -hydroxybenzoic acid, and syringic acid showed KI values in the range of 99,1061 ,m for hCA I and of 105,758 ,m against hCA II, respectively. Quercetin (for hCA I), p -coumaric acid (for hCA II), and gallic acid (for hCA II) exhibited competitive inhibitory effects with 4-nitrophenyl acetate as substrate. All of the other phenolic acids were found as non-competitive inhibitors with 4-nitrophenylacetate as substrate for hCA I and hCA II. The phenolic acids investigated here showed thus interesting hCA I and hCA II inhibitory effects and might be used as leads for generating enzyme inhibitors possibly targeting other CA isoforms which have not been yet assayed for their interactions with such agents. [source] | ||||||||||