scholarly journals Probing the Surface of Human Carbonic Anhydrase for Clues towards the Design of Isoform Specific Inhibitors

2015 ◽  
Vol 2015 ◽  
pp. 1-15 ◽  
Author(s):  
Melissa A. Pinard ◽  
Brian Mahon ◽  
Robert McKenna
Keyword(s):  
Active Site ◽  
Expression Patterns ◽  
Carbonic Anhydrases ◽  
Zinc Metalloenzymes ◽  
High Amino Acid ◽  
Catalytically Active ◽  
Reversible Hydration ◽  
High Amino Acid Conservation ◽  
Specific Inhibitors ◽  
Amino Acid Conservation

The alpha carbonic anhydrases (α-CAs) are a group of structurally related zinc metalloenzymes that catalyze the reversible hydration of CO2toHCO3-. Humans have 15 differentα-CAs with numerous physiological roles and expression patterns. Of these, 12 are catalytically active, and abnormal expression and activities are linked with various diseases, including glaucoma and cancer. Hence there is a need for CA isoform specific inhibitors to avoid off-target CA inhibition, but due to the high amino acid conservation of the active site and surrounding regions between each enzyme, this has proven difficult. However, residues towards the exit of the active site are variable and can be exploited to design isoform selective inhibitors. Here we discuss and characterize this region of “selective drug targetability” and how these observations can be utilized to develop isoform selective CA inhibitors.

scholarly journals Tracking solvent and protein movement during CO2 release in carbonic anhydrase II crystals

2016 ◽  
Vol 113 (19) ◽  
pp. 5257-5262 ◽  
Author(s):  
Chae Un Kim ◽  
HyoJin Song ◽  
Balendu Sankara Avvaru ◽  
Sol M. Gruner ◽  
SangYoun Park ◽  
...  
Keyword(s):  
Carbonic Anhydrase ◽  
Active Site ◽  
Zinc Ion ◽  
Intermediate Water ◽  
Carbonic Anhydrases ◽  
Zinc Metalloenzymes ◽  
Close Proximity ◽  
First Time ◽  
Reversible Hydration ◽  
Co2 Release

Carbonic anhydrases are mostly zinc metalloenzymes that catalyze the reversible hydration/dehydration of CO2/HCO3−. Previously, the X-ray crystal structures of CO2-bound holo (zinc-bound) and apo (zinc-free) human carbonic anhydrase IIs (hCA IIs) were captured at high resolution. Here, we present sequential timeframe structures of holo- [T = 0 s (CO2-bound), 50 s, 3 min, 10 min, 25 min, and 1 h] and apo-hCA IIs [T = 0 s, 50 s, 3 min, and 10 min] during the “slow” release of CO2. Two active site waters, WDW (deep water) and WDW′ (this study), replace the vacated space created on CO2 release, and another water, WI (intermediate water), is seen to translocate to the proton wire position W1. In addition, on the rim of the active site pocket, a water W2′ (this study), in close proximity to residue His64 and W2, gradually exits the active site, whereas His64 concurrently rotates from pointing away (“out”) to pointing toward (“in”) active site rotameric conformation. This study provides for the first time, to our knowledge, structural “snapshots” of hCA II intermediate states during the formation of the His64-mediated proton wire that is induced as CO2 is released. Comparison of the holo- and apo-hCA II structures shows that the solvent network rearrangements require the presence of the zinc ion.

scholarly journals Structural elucidation of the hormonal inhibition mechanism of the bile acid cholate on human carbonic anhydrase II

2014 ◽  
Vol 70 (6) ◽  
pp. 1758-1763 ◽  
Author(s):  
Christopher D. Boone ◽  
Chingkuang Tu ◽  
Robert McKenna
Keyword(s):  
Bile Acid ◽  
Mucosal Injury ◽  
Inhibition Mechanism ◽  
Crystallographic Structure ◽  
Carbonic Anhydrases ◽  
Hydrogen Bond Interactions ◽  
Zinc Metalloenzymes ◽  
Ordered Water ◽  
Biophysical Interactions ◽  
Reversible Hydration

The carbonic anhydrases (CAs) are a family of mostly zinc metalloenzymes that catalyze the reversible hydration/dehydration of CO2into 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 IIviathe 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.

scholarly journals Effect of Sulfonamides and Their Structurally Related Derivatives on the Activity of ι-Carbonic Anhydrase from Burkholderia territorii

2021 ◽  
Vol 22 (2) ◽  
pp. 571
Author(s):  
Viviana De Luca ◽  
Andrea Petreni ◽  
Alessio Nocentini ◽  
Andrea Scaloni ◽  
Claudiu T. Supuran ◽  
...  
Keyword(s):  
Carbon Dioxide ◽  
Antibiotic Resistance ◽  
Pathogenic Bacteria ◽  
Selective Inhibition ◽  
Extensive Study ◽  
Protein Isoforms ◽  
Carbonic Anhydrases ◽  
Physiological Processes ◽  
Microbial Survival ◽  
Reversible Hydration

Carbonic anhydrases (CAs) are essential metalloenzymes in nature, catalyzing the carbon dioxide reversible hydration into bicarbonate and proton. In humans, breathing and many other critical physiological processes depend on this enzymatic activity. The CA superfamily function and inhibition in pathogenic bacteria has recently been the object of significant advances, being demonstrated to affect microbial survival/virulence. Targeting bacterial CAs may thus be a valid alternative to expand the pharmacological arsenal against the emergence of widespread antibiotic resistance. Here, we report an extensive study on the inhibition profile of the recently discovered ι-CA class present in some bacteria, including Burkholderia territorii, namely BteCAι, using substituted benzene-sulfonamides and clinically licensed sulfonamide-, sulfamate- and sulfamide-type drugs. The BteCAι inhibition profile showed: (i) several benzene-sulfonamides with an inhibition constant lower than 100 nM; (ii) a different behavior with respect to other α, β and γ-CAs; (iii) clinically used drugs having a micromolar affinity. This prototype study contributes to the initial recognition of compounds which efficiently and selectively inhibit a bacterial member of the ι-CA class, for which such a selective inhibition with respect to other protein isoforms present in the host is highly desired and may contribute to the development of novel antimicrobials.

scholarly journals Serine protease dynamics revealed by NMR analysis of the thrombin-thrombomodulin complex

2021 ◽  
Vol 11 (1) ◽  
Author(s):  
Riley B. Peacock ◽  
Taylor McGrann ◽  
Marco Tonelli ◽  
Elizabeth A. Komives
Keyword(s):  
Active Site ◽  
Serine Proteases ◽  
Protein C ◽  
Catalytic Mechanism ◽  
Bond Cleavage ◽  
Peptide Bond ◽  
Peptide Bond Cleavage ◽  
Exchange Mass Spectrometry ◽  
Catalytically Active ◽  
Hydrogen Deuterium

AbstractSerine proteases catalyze a multi-step covalent catalytic mechanism of peptide bond cleavage. It has long been assumed that serine proteases including thrombin carry-out catalysis without significant conformational rearrangement of their stable two-β-barrel structure. We present nuclear magnetic resonance (NMR) and hydrogen deuterium exchange mass spectrometry (HDX-MS) experiments on the thrombin-thrombomodulin (TM) complex. Thrombin promotes procoagulative fibrinogen cleavage when fibrinogen engages both the anion binding exosite 1 (ABE1) and the active site. It is thought that TM promotes cleavage of protein C by engaging ABE1 in a similar manner as fibrinogen. Thus, the thrombin-TM complex may represent the catalytically active, ABE1-engaged thrombin. Compared to apo- and active site inhibited-thrombin, we show that thrombin-TM has reduced μs-ms dynamics in the substrate binding (S1) pocket consistent with its known acceleration of protein C binding. Thrombin-TM has increased μs-ms dynamics in a β-strand connecting the TM binding site to the catalytic aspartate. Finally, thrombin-TM had doublet peaks indicative of dynamics that are slow on the NMR timescale in residues along the interface between the two β-barrels. Such dynamics may be responsible for facilitating the N-terminal product release and water molecule entry that are required for hydrolysis of the acyl-enzyme intermediate.

Catalytically Active Site Identification of Molybdenum Disulfide as Gas Cathode in a Nonaqueous Li–CO2 Battery

2021 ◽  
Vol 13 (5) ◽  
pp. 6156-6167
Author(s):  
Chih-Jung Chen ◽  
Chih-Sheng Huang ◽  
Yu-Cheng Huang ◽  
Fu-Ming Wang ◽  
Xing-Chun Wang ◽  
...  
Keyword(s):  
Molybdenum Disulfide ◽  
Active Site ◽  
Catalytically Active ◽  
Site Identification

scholarly journals Expression Patterns and Subcellular Localization of Carbonic Anhydrases Are Developmentally Regulated during Tooth Formation

PLoS ONE ◽  
2014 ◽  
Vol 9 (5) ◽  
pp. e96007 ◽  
Author(s):  
Claes-Göran Reibring ◽  
Maha El Shahawy ◽  
Kristina Hallberg ◽  
Marie Kannius-Janson ◽  
Jeanette Nilsson ◽  
...  
Keyword(s):  
Subcellular Localization ◽  
Expression Patterns ◽  
Carbonic Anhydrases ◽  
Developmentally Regulated ◽  
Tooth Formation

Systematic analysis and integrative discovery of active-site subpocket-specific dehydroquinate synthase inhibitors combating antibiotic-resistant Staphylococcus aureus infection

2018 ◽  
Vol 16 (06) ◽  
pp. 1850027
Author(s):  
Quanfeng Liu ◽  
Liping Li ◽  
Fei Xu
Keyword(s):  
Staphylococcus Aureus ◽  
Active Site ◽  
Shikimate Pathway ◽  
In Vitro Susceptibility ◽  
Systematic Analysis ◽  
Antibiotic Resistant ◽  
Enzyme Active Site ◽  
Specific Inhibitors ◽  
Dehydroquinate Synthase

Shikimate pathway plays an essential role in the biosynthesis of aromatic amino acids in various plants and bacteria, which consists of seven key enzymes and they are all attractive targets for antibacterial agent development due to their absence in humans. The Staphylococcus aureus dehydroquinate synthase (SaDHQS) is involved in the second step of shikimate pathway, which catalyzes the NAD[Formula: see text]-dependent conversion of 3-deoxy-D-arabino-heptulosonate-7-phosphate to dehydroquinate via multiple steps. The enzyme active site can be characterized by two spatially separated subpockets 1 and 2, which represent the reaction center of substrate adduct with NAD[Formula: see text] nicotinamide moiety and the assistant binding site of NAD[Formula: see text] adenine moiety, respectively. In silico virtual screening is performed against a biogenic compound library to discover SaDHQS subpocket-specific inhibitors, which were then tested against both antibiotic-sensitive and antibiotic-resistant S. aureus strains by using in vitro susceptibility test. The activity profile of hit compounds has no considerable difference between the antibiotic-sensitive and -resistant strains. The subpocket 1-specific inhibitors exhibit a generally higher activity than subpocket 2-specific inhibitors, and they also hold a strong selectivity between their cognate and noncognate subpockets. Dynamics and energetics analyses reveal that the SaDHQS active site prefers to interact with amphipathic and polar inhibitors by forming multiple hydrogen bonds and van der Waals packing at the complex interfaces of the two subpockets with their cognate inhibitors.

scholarly journals A validated collection of mouse monoclonal antibodies to human glycosyltransferases functioning in mucin-type O-glycosylation

Glycobiology ◽  
2019 ◽  
Vol 29 (9) ◽  
pp. 645-656 ◽  
Author(s):  
Catharina Steentoft ◽  
Zhang Yang ◽  
Shengjun Wang ◽  
Tongzhong Ju ◽  
Malene B Vester-Christensen ◽  
...  
Keyword(s):  
Monoclonal Antibodies ◽  
Secretory Pathway ◽  
Expression Patterns ◽  
In Situ Monitoring ◽  
Kinetic Properties ◽  
Detailed Knowledge ◽  
Cellular Expression ◽  
Wide Range ◽  
Catalytically Active ◽  
In Cells

Abstract Complex carbohydrates serve a wide range of biological functions in cells and tissues, and their biosynthesis involves more than 200 distinct glycosyltransferases (GTfs) in human cells. The kinetic properties, cellular expression patterns and subcellular topology of the GTfs direct the glycosylation capacity of a cell. Most GTfs are ER or Golgi resident enzymes, and their specific subcellular localization is believed to be distributed in the secretory pathway according to their sequential role in the glycosylation process, although detailed knowledge for individual enzymes is still highly fragmented. Progress in quantitative transcriptome and proteome analyses has greatly advanced our understanding of the cellular expression of this class of enzymes, but availability of appropriate antibodies for in situ monitoring of expression and subcellular topology have generally been limited. We have previously used catalytically active GTfs produced as recombinant truncated secreted proteins in insect cells for generation of mouse monoclonal antibodies (mAbs) to human enzymes primarily involved in mucin-type O-glycosylation. These mAbs can be used to probe subcellular topology of active GTfs in cells and tissues as well as their presence in body fluids. Here, we present several new mAbs to human GTfs and provide a summary of our entire collection of mAbs, available to the community. Moreover, we present validation of specificity for many of our mAbs using human cell lines with CRISPR/Cas9 or zinc finger nuclease (ZFN) knockout and knockin of relevant GTfs.

Mechanism of protein-guided folding of the active site U2/U6 RNA during spliceosome activation

Science ◽  
2020 ◽  
pp. eabc3753
Author(s):  
Cole Townsend ◽  
Majety N. Leelaram ◽  
Dmitry E. Agafonov ◽  
Olexandr Dybkov ◽  
Cindy L. Will ◽  
...  
Keyword(s):  
Active Site ◽  
Rna Structure ◽  
Catalytic Rna ◽  
Folding Pathway ◽  
Correct Order ◽  
Cryo Electron Microscopy ◽  
Assembly Pathway ◽  
Catalytically Active ◽  
Spliceosome Activation ◽  
U6 Rna

Spliceosome activation involves extensive protein and RNA rearrangements that lead to formation of a catalytically-active U2/U6 RNA structure. At present, little is known about the assembly pathway of the latter and the mechanism whereby proteins aid its proper folding. Here we report the cryo-electron microscopy structures of two human pre-Bact complexes at core resolutions of 3.9-4.2 Å. These structures elucidate the order of the numerous protein exchanges that occur during activation, the mutually-exclusive interactions that ensure the correct order of ribonucleoprotein rearrangements needed to form the U2/U6 catalytic RNA, and the stepwise folding pathway of the latter. Structural comparisons with mature Bact complexes reveal the molecular mechanism whereby a conformational change in the scaffold protein PRP8 facilitates final 3D folding of the U2/U6 catalytic RNA.

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