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Mixed Inhibition of cPEPCK by Genistein, Using an Extended Binding Site Located Adjacent to Its Catalytic Cleft.

Katiyar SP, Jain A, Dhanjal JK, Sundar D - PLoS ONE (2015)

Bottom Line: Binding of genistein was also compared with an already known cPEPCK inhibitor.We analyzed the interactions of genistein with cPEPCK enzyme and compared them with its natural substrate (OAA), product (PEP) and known inhibitor (3-MPA).We demonstrate that extended binding site of cPEPCK can further be exploited for designing new drugs against cPEPCK.

View Article: PubMed Central - PubMed

Affiliation: Department of Biochemical Engineering and Biotechnology, Indian Institute of Technology (IIT) Delhi, Hauz Khas, New Delhi, India.

ABSTRACT
Cytosolic phosphoenolpyruvate carboxykinase (cPEPCK) is a critical enzyme involved in gluconeogenesis, glyceroneogenesis and cataplerosis. cPEPCK converts oxaloacetic acid (OAA) into phosphoenol pyruvate (PEP) in the presence of GTP. cPEPCK is known to be associated with type 2 diabetes. Genistein is an isoflavone compound that shows anti-diabetic and anti-obesitic properties. Experimental studies have shown a decrease in the blood glucose level in the presence of genistein by lowering the functional activity of cPEPCK, an enzyme of gluconeogenesis. Using computational techniques such as molecular modeling, molecular docking, molecular dynamics simulation and binding free energy calculations, we identified cPEPCK as a direct target of genistein. We studied the molecular interactions of genistein with three possible conformations of cPEPCK-unbound cPEPCK (u_cPEPCK), GTP bound cPEPCK (GTP_cPEPCK) and GDP bound cPEPCK (GDP_cPEPCK). Binding of genistein was also compared with an already known cPEPCK inhibitor. We analyzed the interactions of genistein with cPEPCK enzyme and compared them with its natural substrate (OAA), product (PEP) and known inhibitor (3-MPA). Our results demonstrate that genistein uses the mechanism of mixed inhibition to block the functional activity of cPEPCK and thus can serve as a potential anti-diabetic and anti-obesity drug candidate. We also identified an extended binding site in the catalytic cleft of cPEPCK which is used by 3-MPA to inhibit cPEPCK non-competitively. We demonstrate that extended binding site of cPEPCK can further be exploited for designing new drugs against cPEPCK.

No MeSH data available.


Related in: MedlinePlus

Fluctuation in residue Asn292 and Asp310 during the MD simulations.Changeability of GTP interacting residues indicates the change in position of ligand with respect to GTP binding site during the MD simulation. A) RMSD of residue Asn292 in refined cPEPCK protein backbone (black), backbone of u_cPEPCK-genistein complex (red), backbone of GTP_cPEPCK-genistein complex (blue), backbone of GDP_cPEPCK-genistein complex (yellow), and backbone of OAA-GTP_cPEPCK protein-ligand complex. B) RMSD of residue Asp310 in refined cPEPCK protein backbone (black), backbone of u_cPEPCK-genistein complex (red), backbone of GTP_cPEPCK-genistein complex (blue), backbone of GDP_cPEPCK-genistein complex (yellow), and backbone of OAA-GTP_cPEPCK protein-ligand complex.
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pone.0141987.g002: Fluctuation in residue Asn292 and Asp310 during the MD simulations.Changeability of GTP interacting residues indicates the change in position of ligand with respect to GTP binding site during the MD simulation. A) RMSD of residue Asn292 in refined cPEPCK protein backbone (black), backbone of u_cPEPCK-genistein complex (red), backbone of GTP_cPEPCK-genistein complex (blue), backbone of GDP_cPEPCK-genistein complex (yellow), and backbone of OAA-GTP_cPEPCK protein-ligand complex. B) RMSD of residue Asp310 in refined cPEPCK protein backbone (black), backbone of u_cPEPCK-genistein complex (red), backbone of GTP_cPEPCK-genistein complex (blue), backbone of GDP_cPEPCK-genistein complex (yellow), and backbone of OAA-GTP_cPEPCK protein-ligand complex.

Mentions: Binding site of cPEPCK is a large cavity comprising of a GTP/GDP binding site and a substrate/product binding pocket (S2 Fig). Binding site of cPEPCK covers an area of 2754.15 Å2, out of which 1789.55 Å2 is hydrophilic and 350 Å2 is of hydrophobic in nature. High hydrophilic nature of the binding site is justified as it has to accommodate GDP/GTP cofactor that contains highly negatively charged phosphate groups. Such large binding pocket can accommodate a cofactor such as GTP that has surface area of approximately 400 Å2 and a ligand such as genistein that has surface area of approximately 250 Å2 in bound conformation. Fluctuations of binding site residues were recorded during the MD simulation of u_cPEPCK protein. Although most of the residues lining the binding pocket did not show much flexibility, Asn292 and Asp310 showed high fluctuations indicating that their side chains were free to move within the binding cavity (Fig 2A and 2B). These residues were located near the GTP/GDP binding site and play an important role by interacting with GTP/GDP. Binding site analysis of a cPEPCK structure from stable trajectory reveals that the binding site expanded and merged with a neighboring cavity, which was adjacent to GTP and substrate/product binding pocket. The area of this extended binding cavity becomes 4184.51 Å2, out of which 2865 Å2 forms the hydrophilic surface. Role of the adjacent cavity is described later in this study.


Mixed Inhibition of cPEPCK by Genistein, Using an Extended Binding Site Located Adjacent to Its Catalytic Cleft.

Katiyar SP, Jain A, Dhanjal JK, Sundar D - PLoS ONE (2015)

Fluctuation in residue Asn292 and Asp310 during the MD simulations.Changeability of GTP interacting residues indicates the change in position of ligand with respect to GTP binding site during the MD simulation. A) RMSD of residue Asn292 in refined cPEPCK protein backbone (black), backbone of u_cPEPCK-genistein complex (red), backbone of GTP_cPEPCK-genistein complex (blue), backbone of GDP_cPEPCK-genistein complex (yellow), and backbone of OAA-GTP_cPEPCK protein-ligand complex. B) RMSD of residue Asp310 in refined cPEPCK protein backbone (black), backbone of u_cPEPCK-genistein complex (red), backbone of GTP_cPEPCK-genistein complex (blue), backbone of GDP_cPEPCK-genistein complex (yellow), and backbone of OAA-GTP_cPEPCK protein-ligand complex.
© Copyright Policy
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4631375&req=5

pone.0141987.g002: Fluctuation in residue Asn292 and Asp310 during the MD simulations.Changeability of GTP interacting residues indicates the change in position of ligand with respect to GTP binding site during the MD simulation. A) RMSD of residue Asn292 in refined cPEPCK protein backbone (black), backbone of u_cPEPCK-genistein complex (red), backbone of GTP_cPEPCK-genistein complex (blue), backbone of GDP_cPEPCK-genistein complex (yellow), and backbone of OAA-GTP_cPEPCK protein-ligand complex. B) RMSD of residue Asp310 in refined cPEPCK protein backbone (black), backbone of u_cPEPCK-genistein complex (red), backbone of GTP_cPEPCK-genistein complex (blue), backbone of GDP_cPEPCK-genistein complex (yellow), and backbone of OAA-GTP_cPEPCK protein-ligand complex.
Mentions: Binding site of cPEPCK is a large cavity comprising of a GTP/GDP binding site and a substrate/product binding pocket (S2 Fig). Binding site of cPEPCK covers an area of 2754.15 Å2, out of which 1789.55 Å2 is hydrophilic and 350 Å2 is of hydrophobic in nature. High hydrophilic nature of the binding site is justified as it has to accommodate GDP/GTP cofactor that contains highly negatively charged phosphate groups. Such large binding pocket can accommodate a cofactor such as GTP that has surface area of approximately 400 Å2 and a ligand such as genistein that has surface area of approximately 250 Å2 in bound conformation. Fluctuations of binding site residues were recorded during the MD simulation of u_cPEPCK protein. Although most of the residues lining the binding pocket did not show much flexibility, Asn292 and Asp310 showed high fluctuations indicating that their side chains were free to move within the binding cavity (Fig 2A and 2B). These residues were located near the GTP/GDP binding site and play an important role by interacting with GTP/GDP. Binding site analysis of a cPEPCK structure from stable trajectory reveals that the binding site expanded and merged with a neighboring cavity, which was adjacent to GTP and substrate/product binding pocket. The area of this extended binding cavity becomes 4184.51 Å2, out of which 2865 Å2 forms the hydrophilic surface. Role of the adjacent cavity is described later in this study.

Bottom Line: Binding of genistein was also compared with an already known cPEPCK inhibitor.We analyzed the interactions of genistein with cPEPCK enzyme and compared them with its natural substrate (OAA), product (PEP) and known inhibitor (3-MPA).We demonstrate that extended binding site of cPEPCK can further be exploited for designing new drugs against cPEPCK.

View Article: PubMed Central - PubMed

Affiliation: Department of Biochemical Engineering and Biotechnology, Indian Institute of Technology (IIT) Delhi, Hauz Khas, New Delhi, India.

ABSTRACT
Cytosolic phosphoenolpyruvate carboxykinase (cPEPCK) is a critical enzyme involved in gluconeogenesis, glyceroneogenesis and cataplerosis. cPEPCK converts oxaloacetic acid (OAA) into phosphoenol pyruvate (PEP) in the presence of GTP. cPEPCK is known to be associated with type 2 diabetes. Genistein is an isoflavone compound that shows anti-diabetic and anti-obesitic properties. Experimental studies have shown a decrease in the blood glucose level in the presence of genistein by lowering the functional activity of cPEPCK, an enzyme of gluconeogenesis. Using computational techniques such as molecular modeling, molecular docking, molecular dynamics simulation and binding free energy calculations, we identified cPEPCK as a direct target of genistein. We studied the molecular interactions of genistein with three possible conformations of cPEPCK-unbound cPEPCK (u_cPEPCK), GTP bound cPEPCK (GTP_cPEPCK) and GDP bound cPEPCK (GDP_cPEPCK). Binding of genistein was also compared with an already known cPEPCK inhibitor. We analyzed the interactions of genistein with cPEPCK enzyme and compared them with its natural substrate (OAA), product (PEP) and known inhibitor (3-MPA). Our results demonstrate that genistein uses the mechanism of mixed inhibition to block the functional activity of cPEPCK and thus can serve as a potential anti-diabetic and anti-obesity drug candidate. We also identified an extended binding site in the catalytic cleft of cPEPCK which is used by 3-MPA to inhibit cPEPCK non-competitively. We demonstrate that extended binding site of cPEPCK can further be exploited for designing new drugs against cPEPCK.

No MeSH data available.


Related in: MedlinePlus