Possible Roles for His 208 in the Active-Site Region of Chloroplast Carbonic Anhydrase fromPisum sativum

To find novel human carbonic anhydrase (hCA) inhibitors, we synthesized thirteen compounds by combining thiazolidinone with benzenesulfonamide. The result of the X-ray single-crystal diffraction experiment confirmed the configuration of this class of compounds. The enzyme inhibition assays against hCA II and IX showed desirable potency profiles, as effective as the positive controls. The docking studies revealed that compounds (2) and (7) efficiently bound in the active site cavity of hCA IX by forming sufficient interactions with active site residues. The fragment of thiazolidinone played an important role in the binding of the molecules to the active site.

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Faculty Opinions recommendation of Active-site solvent replenishment observed during human carbonic anhydrase II catalysis.

Faculty Opinions – Post-Publication Peer Review of the Biomedical Literature ◽

10.3410/f.732394273.793552562 ◽

2018 ◽

Author(s):

Alberto Podjarny

Keyword(s):

Carbonic Anhydrase ◽

Active Site ◽

Carbonic Anhydrase Ii ◽

Human Carbonic Anhydrase

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A crystallographic study of the Toho-1 β-lactamase acylation mechanism

Acta Crystallographica Section A Foundations and Advances ◽

10.1107/s2053273314087920 ◽

2014 ◽

Vol 70 (a1) ◽

pp. C1207-C1207

Author(s):

Leighton Coates

Keyword(s):

Hydrogen Bonding ◽

Transition State ◽

Active Site ◽

Catalytic Mechanism ◽

Substrate Binding ◽

Ligand Complex ◽

Transition State Analog ◽

Hydrogen Bonding Interactions ◽

Active Site Region ◽

Neutron Crystallography

β-lactam antibiotics have been used effectively over several decades against many types of highly virulent bacteria. The predominant cause of resistance to these antibiotics in Gram-negative bacterial pathogens is the production of serine β-lactamase enzymes. A key aspect of the class A serine β-lactamase mechanism that remains unresolved and controversial is the identity of the residue acting as the catalytic base during the acylation reaction. Multiple mechanisms have been proposed for the formation of the acyl-enzyme intermediate that are predicated on understanding the protonation states and hydrogen-bonding interactions among the important residues involved in substrate binding and catalysis of these enzymes. For resolving a controversy of this nature surrounding the catalytic mechanism, neutron crystallography is a powerful complement to X-ray crystallography that can explicitly determine the location of deuterium atoms in proteins, thereby directly revealing the hydrogen-bonding interactions of important amino acid residues. Neutron crystallography was used to unambiguously reveal the ground-state active site protonation states and the resulting hydrogen-bonding network in two ligand-free Toho-1 β-lactamase mutants which provided remarkably clear pictures of the active site region prior to substrate binding and subsequent acylation [1,2] and an acylation transition-state analog, benzothiophene-2-boronic acid (BZB), which was also isotopically enriched with 11B. The neutron structure revealed the locations of all deuterium atoms in the active site region and clearly indicated that Glu166 is protonated in the BZB transition-state analog complex. As a result, the complete hydrogen-bonding pathway throughout the active site region could then deduced for this protein-ligand complex that mimics the acylation tetrahedral intermediate [3].

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Structural insights into the effect of active-site mutation on the catalytic mechanism of carbonic anhydrase

IUCrJ ◽

10.1107/s2052252520011008 ◽

2020 ◽

Vol 7 (6) ◽

pp. 985-994 ◽

Cited By ~ 1

Author(s):

Jin Kyun Kim ◽

Cheol Lee ◽

Seon Woo Lim ◽

Jacob T. Andring ◽

Aniruddha Adhikari ◽

...

Keyword(s):

Carbonic Anhydrase ◽

Kinetic Parameters ◽

Active Site ◽

Systematic Approach ◽

Catalytic Mechanism ◽

Structural Information ◽

Site Directed Mutagenesis ◽

Site Mutation ◽

Functional Changes ◽

Reaction Rate Constants

Enzymes are catalysts of biological processes. Significant insight into their catalytic mechanisms has been obtained by relating site-directed mutagenesis studies to kinetic activity assays. However, revealing the detailed relationship between structural modifications and functional changes remains challenging owing to the lack of information on reaction intermediates and of a systematic way of connecting them to the measured kinetic parameters. Here, a systematic approach to investigate the effect of an active-site-residue mutation on a model enzyme, human carbonic anhydrase II (CA II), is described. Firstly, structural analysis is performed on the crystallographic intermediate states of native CA II and its V143I variant. The structural comparison shows that the binding affinities and configurations of the substrate (CO2) and product (HCO3 −) are altered in the V143I variant and the water network in the water-replenishment pathway is restructured, while the proton-transfer pathway remains mostly unaffected. This structural information is then used to estimate the modifications of the reaction rate constants and the corresponding free-energy profiles of CA II catalysis. Finally, the obtained results are used to reveal the effect of the V143I mutation on the measured kinetic parameters (k cat and k cat/K m) at the atomic level. It is believed that the systematic approach outlined in this study may be used as a template to unravel the structure–function relationships of many other biologically important enzymes.

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