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Title: Structural elucidation of the hormonal inhibition mechanism of the bile acid cholate on human carbonic anhydrase II

Abstract

The structure of human carbonic anhydrase II in complex with cholate has been determined to 1.54 Å resolution. Elucidation of the novel inhibition mechanism of cholate will aid in the development of a nonsulfur-containing, isoform-specific therapeutic agent. The carbonic anhydrases (CAs) are a family of mostly zinc metalloenzymes that catalyze the reversible hydration/dehydration of CO{sub 2} into bicarbonate and a proton. Human isoform CA II (HCA II) is abundant in the surface epithelial cells of the gastric mucosa, where it serves an important role in cytoprotection through bicarbonate secretion. Physiological inhibition of HCA II via the bile acids contributes to mucosal injury in ulcerogenic conditions. This study details the weak biophysical interactions associated with the binding of a primary bile acid, cholate, to HCA II. The X-ray crystallographic structure determined to 1.54 Å resolution revealed that cholate does not make any direct hydrogen-bond interactions with HCA II, but instead reconfigures the well ordered water network within the active site to promote indirect binding to the enzyme. Structural knowledge of the binding interactions of this nonsulfur-containing inhibitor with HCA II could provide the template design for high-affinity, isoform-specific therapeutic agents for a variety of diseases/pathological states, including cancer, glaucoma, epilepsy andmore » osteoporosis.« less

Authors:
 [1];  [2];  [1]
  1. University of Florida, PO Box 100267, Gainesville, FL 32610 (United States)
  2. University of Florida, PO Box 100245, Gainesville, FL 32610 (United States)
Publication Date:
OSTI Identifier:
22351325
Resource Type:
Journal Article
Resource Relation:
Journal Name: Acta Crystallographica. Section D: Biological Crystallography; Journal Volume: 70; Journal Issue: Pt 6; Other Information: PMCID: PMC4051509; PMID: 24914985; PUBLISHER-ID: rr5068; OAI: oai:pubmedcentral.nih.gov:4051509; Copyright (c) International Union of Crystallography 2014; Country of input: International Atomic Energy Agency (IAEA)
Country of Publication:
Denmark
Language:
English
Subject:
75 CONDENSED MATTER PHYSICS, SUPERCONDUCTIVITY AND SUPERFLUIDITY; AFFINITY; DEHYDRATION; DESIGN; DRUGS; HYDRATION; HYDROGEN; INHIBITION; INTERACTIONS; NEOPLASMS; PROTONS; RESOLUTION; SURFACES; WATER; ZINC

Citation Formats

Boone, Christopher D., Tu, Chingkuang, and McKenna, Robert, E-mail: rmckenna@ufl.edu. Structural elucidation of the hormonal inhibition mechanism of the bile acid cholate on human carbonic anhydrase II. Denmark: N. p., 2014. Web. doi:10.1107/S1399004714007457.
Boone, Christopher D., Tu, Chingkuang, & McKenna, Robert, E-mail: rmckenna@ufl.edu. Structural elucidation of the hormonal inhibition mechanism of the bile acid cholate on human carbonic anhydrase II. Denmark. doi:10.1107/S1399004714007457.
Boone, Christopher D., Tu, Chingkuang, and McKenna, Robert, E-mail: rmckenna@ufl.edu. 2014. "Structural elucidation of the hormonal inhibition mechanism of the bile acid cholate on human carbonic anhydrase II". Denmark. doi:10.1107/S1399004714007457.
@article{osti_22351325,
title = {Structural elucidation of the hormonal inhibition mechanism of the bile acid cholate on human carbonic anhydrase II},
author = {Boone, Christopher D. and Tu, Chingkuang and McKenna, Robert, E-mail: rmckenna@ufl.edu},
abstractNote = {The structure of human carbonic anhydrase II in complex with cholate has been determined to 1.54 Å resolution. Elucidation of the novel inhibition mechanism of cholate will aid in the development of a nonsulfur-containing, isoform-specific therapeutic agent. The carbonic anhydrases (CAs) are a family of mostly zinc metalloenzymes that catalyze the reversible hydration/dehydration of CO{sub 2} into bicarbonate and a proton. Human isoform CA II (HCA II) is abundant in the surface epithelial cells of the gastric mucosa, where it serves an important role in cytoprotection through bicarbonate secretion. Physiological inhibition of HCA II via the bile acids contributes to mucosal injury in ulcerogenic conditions. This study details the weak biophysical interactions associated with the binding of a primary bile acid, cholate, to HCA II. The X-ray crystallographic structure determined to 1.54 Å resolution revealed that cholate does not make any direct hydrogen-bond interactions with HCA II, but instead reconfigures the well ordered water network within the active site to promote indirect binding to the enzyme. Structural knowledge of the binding interactions of this nonsulfur-containing inhibitor with HCA II could provide the template design for high-affinity, isoform-specific therapeutic agents for a variety of diseases/pathological states, including cancer, glaucoma, epilepsy and osteoporosis.},
doi = {10.1107/S1399004714007457},
journal = {Acta Crystallographica. Section D: Biological Crystallography},
number = Pt 6,
volume = 70,
place = {Denmark},
year = 2014,
month = 6
}
  • The X-ray crystallographic structure of the disulfide-containing HCAII (dsHCAII) has been solved to 1.77 Å resolution and revealed that successful oxidation of the cysteine bond was achieved while also retaining desirable active-site geometry. The carbonic anhydrases (CAs) are a family of mostly zinc metalloenzymes that catalyze the reversible hydration of CO{sub 2} to bicarbonate and a proton. Recently, there has been industrial interest in utilizing CAs as biocatalysts for carbon sequestration and biofuel production. The conditions used in these processes, however, result in high temperatures and acidic pH. This unfavorable environment results in rapid destabilization and loss of catalytic activitymore » in CAs, ultimately resulting in cost-inefficient high-maintenance operation of the system. In order to negate these detrimental industrial conditions, cysteines at residues 23 (Ala23Cys) and 203 (Leu203Cys) were engineered into a wild-type variant of human CA II (HCAII) containing the mutation Cys206Ser. The X-ray crystallographic structure of the disulfide-containing HCAII (dsHCAII) was solved to 1.77 Å resolution and revealed that successful oxidation of the cysteine bond was achieved while also retaining desirable active-site geometry. Kinetic studies utilizing the measurement of {sup 18}O-labeled CO{sub 2} by mass spectrometry revealed that dsHCAII retained high catalytic efficiency, and differential scanning calorimetry showed acid stability and thermal stability that was enhanced by up to 14 K compared with native HCAII. Together, these studies have shown that dsHCAII has properties that could be used in an industrial setting to help to lower costs and improve the overall reaction efficiency.« less
  • A detailed analysis of the stability and activity of Mn(II) human carbonic anhydrase I and the kinetics and mechanism of its catalysis of the HCO/sub 3//sup -//CO/sub 2/ exchange have been performed at pH 8.5. The analysis was based on the paramagnetic relaxation rates R/sub 1p/ and R/sub 2p/ of the /sup 13/C atom of HCO/sub 3//sup -/ in the Mn/sup 2 +//apoenzyme/HCO/sub 3//sup -//CO/sub 2/ system and the HCO/sub 3//sup -/ in equilibrium CO/sub 2/ interconversion rate obtained by the magnetization-transfer technique. The R/sub 1p/ and R/sub 2p/ rates were measured as functions of the temperature, magnetic field strength,more » and substrate and apoenzyme concentrations and were interpreted on the basis of Solomon-Bloembergen-Morgan theories and general equations for the liquid exchange. From the analysis of the data, a formation constant for the Mn(II) enzyme of log K/sub MA//sup M/ = 5.8 +/- 0.4 was obtained while the activity of the Mn(II) enzyme, measured as the HCO/sub 3//sup -//CO/sub 2/ interconversion rate at (HCO/sub 3//sup -/) = 0.100 M and pH 8.5, was found to be about 4% of that of the native Zn(II) enzyme. Most conspicuously, the resulting distance of only 2.71 +/- 0.03 A between the Mn/sup 2 +/ ion of the enzyme and the /sup 13/C atom of HCO/sub 3//sup -/ in the enzyme-bicarbonate complex indicates that the bicarbonate is bound to the metal ion by two of its oxygen atoms in the central catalytic step, thereby supporting the modified Zn(II)-OH mechanism. In contrast, this binding mode differs from the structure of the complexes suggested in the rapid-equilibrium kinetic model.« less
  • The catalytic mechanism of human carbonic anhydrase II (HCAII) has been studied by using a combined high-level ab initio and free energy perturbation approach. The authors have examined two hydration reaction mechanisms. The first involves hydration of CO[sub 2] followed by an internal proton transfer and then loss of bicarbonate. They find that the former mechanism is more likely than the latter. On the basis of their results they have been able to predict the location of the bicarbonate proton in the recently solved X-ray structure of the HCAII-bicarbonate complex. They find that the proton is hydrogen bound to Thr-199,more » while one of the bicarbonate oxygens is hydrogen bound to the main-chain NH of Thr-199. This hydrogen-bonding pattern is analogous to that seen for sulfoamides, which suggests that these molecules are substrate (or transition-state) mimics. Their calculated free energy barrier for CO[sub 2] hydration is in reasonable accord with experiment, while that for the dehydration of bicarbonate is in poor agreement. The reasons for this disagreement are discussed. The molecular-level details obtained from this study have been used to construct a detailed catalytic mechanism for the mode of action of HCAII. 59 refs., 9 figs., 2 tabs.« less
  • Many zinc enzymes utilize zinc bound water as a critical component of a catalytic reaction. The Zn2+ ion activates water through ionization, polarization, or simple displacement depending upon the mechanistic details. The fate of one proton from the bound water is determined primarily by the influence of directly bound Zn-ligands, as well as hydrogen bonding with a secondary coordination sphere of side chains and/or bound waters within the protein. We have employed low temperature solid-state 67Zn NMR spectroscopy to probe the nature of the bonding at Zn2+ in human carbonic anhydrase isozyme II (CAII). In particular we wanted to characterizemore » the 67Zn NMR parameters of the metal with both water and hydroxide as the fourth ligand, but instead we show that hydroxide is bound to Zn2+ over the pH range of 5 to 8.5. These results suggest the accepted mechanism of action of CAII needs to be revised. These data serve to provide further understanding of the observed pH dependence of the activity of this well studied protein.« less
  • Recent studies have shown that cadmium inhibits electron transport in isolated chloroplasts. Li and Miles demonstrated that the oxidizing side of PS II is the most sensitive site of inhibition. Concentrations exceeding 0.5 mM are required to cause strong inhibition of electron transport. Our studies on Cd inhibition of electron transport in isolated chloroplast confirm the findings of Li and Miles. In addition we find that Cd strongly inhibits carbonic anhydrase activity at concentrations much lower than those reported for inhibition of electron transport. We obtain strong inhibition of carbonic anhydrase at 0.05 mM Cd Cl/sub 2/. The degree ofmore » inhibition is similar in broken or intact chloroplasts after either light or dark pre-treatments. Other evidence such as the inhibition obtained when chloroplasts are removed from assay solutions containing Cd prior to the enzyme assay shows that Cd acts after rapid penetration through the chloroplast membrane. Since we find that other enzymes such as carboxydismutase involved in the dark reactions are also susceptible to Cd our results are indicative that the CO/sub 2/ assimilation could be more sensitive to Cd inhibition than electron transport.« less