Mutation of active site residues Asn67 to Ile, Gln92 to Val and Leu204 to Ser in human carbonic anhydrase II: Influences on the catalytic activity and affinity for inhibitors

Türkoğlu, Sümeyye Aydoğan; Maresca, Alfonso; Alper, Meltem; Köçkar, Feray; Işık, Semra; Sinan, Selma; Ozensoy, Ozen; Arslan, Oktay; Supuran, Claudiu T.

doi:10.1016/j.bmc.2012.02.029

Cited by 8 publications

(6 citation statements)

References 65 publications

Supporting

Mentioning

Contrasting

Order By: Relevance

“…The earlier work confirmed that specific substitutes of residue in the hydrophobic pocket were related to variations in catalytic rates of hCA II (Fierke et al, 1991;Host and Jonsson, 2008). While the exchanges of Asn67 to Ile, Gln92 to Val, and Leu204 to Ser in the active site of hCA II were carried out by Turkoglu et al (2012), W209I, W209L, W209V, and W209P mutant enzymes were obtained in our study. The mutations at Val 143 at the CO 2 binding site of hCA II lead to important modifications in the three-dimensional protein structure and catalytic activity (Fierke et al, 1991).…”

Section: Discussionsupporting

confidence: 87%

“…Mutants of CA I and CA II have been obtained and enzyme activities of these mutants have been associated with wildtype in many studies. CO 2 and 4-nitrophenylacetate were used as a substrate in these enzyme assays (Nair et al, 1991;Nair and Christianson, 1993;Fisher et al, 2005;Jiang et al, 2008;Kockar et al, 2010;Mikulski et al, 2011aMikulski et al, , 2011bWu et al, 2011;Fisher et al, 2012;Turkoglu et al, 2012;Halder and Taraphder, 2013). The earlier work confirmed that specific substitutes of residue in the hydrophobic pocket were related to variations in catalytic rates of hCA II (Fierke et al, 1991;Host and Jonsson, 2008).…”

Section: Discussionmentioning

confidence: 94%

See 1 more Smart Citation

Effect of mutation in active site residue Trp209 to Val, Leu, Ile and Pro on the catalytic activity and affinity for some benzenesulfonamides of human carbonic anhydrase II

Kılıç¹,

Erdoğan²,

Küfrevioğlu³

2017

Turk J Biol

View full text Add to dashboard Cite

Human carbonic anhydrase II (hCA II) enzyme was firstly expressed using a pET-SUMO expression vector in Escherichia coli and the recombinant enzyme was purified using nickel (Ni 2+ ) affinity chromatography. The substitutions of Trp 209 with four amino acid (Val, Leu, Ile, and Pro) in the hydrophobic pocket of hCA II were conducted using site-directed mutagenesis. The p-nitrophenyl esterase activity of hCA II variants correlates with the hydrophobicity and size of residue, suggesting that the hydrophobic character of this residue is important for catalysis. The Trp 209 was forecast as an important residue and was exposed to computational mutagenesis. This forecast was confirmed experimentally by producing hCA II mutants and determining the resulting affinities towards some benzenesulfonamides. These mutations in the hydrophobic pocket of the enzyme active site decreased the protein expression of hCA II in E. coli, causing the formation of insoluble protein aggregates in many cases. Our findings demonstrated that the Trp 209 in hCA II plays an important role in the folding process and the valine residues are very compatible for the hydrophobic region in the active cavity of this isoenzyme. These mutant proteins will lead to a better understanding of structural functions and drug-based studies in the future.

show abstract

Section: Discussionsupporting

confidence: 87%

Section: Discussionmentioning

confidence: 94%

Effect of mutation in active site residue Trp209 to Val, Leu, Ile and Pro on the catalytic activity and affinity for some benzenesulfonamides of human carbonic anhydrase II

Kılıç¹,

Erdoğan²,

Küfrevioğlu³

2017

Turk J Biol

View full text Add to dashboard Cite

show abstract

“…9 standpoint, compounds containing alkyl chains can establish favorable interactions with the hydrophobic patch at the enzymatic cavity, often referred to as the "hydrophobic wall". 10 The role of these interactions has recently been confirmed by isothermal titration calorimetry (ITC) experiments, 11,12 molecular dynamics (MD), 12 site-directed mutagenesis, 13−15 and X-ray crystallography data, 16−18 which is thoroughly reviewed by De Simone et al 19 Further complexity to the binding mechanism is given by the charge state of the sulfonamide, which binds to the zinc ion in its anionic form, as reported by 15 N NMR 20 and recent neutron diffraction studies. 21 Since benzenesulfonamides are usually weak acids, it is expected that these inhibitors initially bind to hCAII in their neutral form and only subsequently deprotonate upon reaching the final Zn 2+ -coordinated configuration.…”

Section: ■ Introductionmentioning

confidence: 89%

“…From a structural standpoint, compounds containing alkyl chains can establish favorable interactions with the hydrophobic patch at the enzymatic cavity, often referred to as the “hydrophobic wall” . The role of these interactions has recently been confirmed by isothermal titration calorimetry (ITC) experiments, , molecular dynamics (MD), site-directed mutagenesis, − and X-ray crystallography data, − which is thoroughly reviewed by De Simone et al…”

Section: Introductionmentioning

confidence: 99%

Kinetic and Structural Insights into the Mechanism of Binding of Sulfonamides to Human Carbonic Anhydrase by Computational and Experimental Studies

et al. 2016

View full text Add to dashboard Cite

The binding of sulfonamides to human carbonic anhydrase II (hCAII) is a complex and long-debated example of protein-ligand recognition and interaction. In this study, we investigate the para-substituted n-alkyl and hydroxyethylene-benzenesulfonamides, providing a complete reconstruction of their binding pathway to hCAII by means of large-scale molecular dynamics simulations, density functional calculations, surface plasmon resonance (SPR) measurements, and X-ray crystallography experiments. Our analysis shows that the protein-ligand association rate (kon) dramatically increases with the ligand's hydrophobicity, pointing to the existence of a prebinding stage largely stabilized by a favorable packing of the ligand's apolar moieties with the hCAII "hydrophobic wall". The characterization of the binding pathway allows an unprecedented understanding of the structure-kinetic relationship in hCAII/benzenesulfonamide complexes, depicting a paradigmatic scenario for the multistep binding process in protein-ligand systems.

show abstract

“…In order to elucidate the catalytic details of the active site of CA II, several mutational studies have been performed. Most of these variants have been focused at the zinc ion-binding site (Alexander et al, 1993;Kiefer et al, 1993;Ippolito & Christianson, 1994;Lesburg & Christianson, 1995;Huang et al, 1996;Lesburg et al, 1997), the hydrophilic side (protontransfer pathway; Behravan et al, 1990;Krebs, Ippolito et al, 1993;Xue et al, 1993;Ippolito et al, 1995;Huang et al, 2002;Tu et al, 2002;Fisher et al, 2005;Zheng et al, 2008;Turkoglu et al, 2012;Mikulski et al, 2013;Aggarwal et al, 2014) and the hydrophobic pocket (CO 2 -binding site; Alexander et al, 1991;Fierke et al, 1991;Krebs, Rana et al, 1993;West et al, 2012;Nair & Christianson, 1993). In the hydrophobic pocket, a series of mutational studies have been performed targeting the Val121, Val143 and Leu198 residues.…”

Section: Introductionmentioning

confidence: 99%

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

Kim

Lee

Lim

et al. 2020

Int Union Crystallogr J

View full text Add to dashboard Cite

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.

show abstract

Mutation of active site residues Asn67 to Ile, Gln92 to Val and Leu204 to Ser in human carbonic anhydrase II: Influences on the catalytic activity and affinity for inhibitors

Cited by 8 publications

References 65 publications

Effect of mutation in active site residue Trp209 to Val, Leu, Ile and Pro on the catalytic activity and affinity for some benzenesulfonamides of human carbonic anhydrase II

Effect of mutation in active site residue Trp209 to Val, Leu, Ile and Pro on the catalytic activity and affinity for some benzenesulfonamides of human carbonic anhydrase II

Kinetic and Structural Insights into the Mechanism of Binding of Sulfonamides to Human Carbonic Anhydrase by Computational and Experimental Studies

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

Contact Info

Product

Resources

About