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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

Change of binding site by both genistein and OAA after the MD simulations.Superimposed pre MD simulation and post MD simulation structures of OAA-GTP_cPEPCK and genistein-GTP_cPEPCK complexes, revealing the presence of possible binding sites within cPEPCK. Before the 30 ns MD simulation, OAA (light red) and genistein (light blue) are located at the same site while after the MD simulation OAA (dark red) and genistein (dark blue), both are located at the same extended binding site.
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pone.0141987.g006: Change of binding site by both genistein and OAA after the MD simulations.Superimposed pre MD simulation and post MD simulation structures of OAA-GTP_cPEPCK and genistein-GTP_cPEPCK complexes, revealing the presence of possible binding sites within cPEPCK. Before the 30 ns MD simulation, OAA (light red) and genistein (light blue) are located at the same site while after the MD simulation OAA (dark red) and genistein (dark blue), both are located at the same extended binding site.

Mentions: The changes in the conformation of active site in GTP_cPEPCK-gensitein complex before and after MD are shown in Fig 5C. Genistein was found trapped into an alternate small binding site located just adjacent to the main binding site (Fig 6). Glu86 and Cys245 were the residues interacting with genistein at the extended binding site of GTP_cPEPCK (Table 2 and S3D Fig). Also the distance analysis between genistein and Arg87, Tyr235, Arg249 suggested that in the presence of GTP, genistein stabilizes itself near the substrate/product binding site by moving into an extended binding site (Figure 3 in S1 Appendix). This indicates that behavior of genistein at the cPEPCK binding is different in the presence of GTP than that in unbound cPEPCK. In the presence of GTP, genistein stabilizes itself at the substrate/product binding site as opposed to moving towards GTP binding site in absence of any cofactor at cPEPCK binding site. Binding free energy (ΔG) of genistein with GTP_cPEPCK was estimated to be -23.26/-16.75 kcal/mol(±3.42) using MMGB/PBSA method (Table 1).


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)

Change of binding site by both genistein and OAA after the MD simulations.Superimposed pre MD simulation and post MD simulation structures of OAA-GTP_cPEPCK and genistein-GTP_cPEPCK complexes, revealing the presence of possible binding sites within cPEPCK. Before the 30 ns MD simulation, OAA (light red) and genistein (light blue) are located at the same site while after the MD simulation OAA (dark red) and genistein (dark blue), both are located at the same extended binding site.
© Copyright Policy
Related In: Results  -  Collection

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

pone.0141987.g006: Change of binding site by both genistein and OAA after the MD simulations.Superimposed pre MD simulation and post MD simulation structures of OAA-GTP_cPEPCK and genistein-GTP_cPEPCK complexes, revealing the presence of possible binding sites within cPEPCK. Before the 30 ns MD simulation, OAA (light red) and genistein (light blue) are located at the same site while after the MD simulation OAA (dark red) and genistein (dark blue), both are located at the same extended binding site.
Mentions: The changes in the conformation of active site in GTP_cPEPCK-gensitein complex before and after MD are shown in Fig 5C. Genistein was found trapped into an alternate small binding site located just adjacent to the main binding site (Fig 6). Glu86 and Cys245 were the residues interacting with genistein at the extended binding site of GTP_cPEPCK (Table 2 and S3D Fig). Also the distance analysis between genistein and Arg87, Tyr235, Arg249 suggested that in the presence of GTP, genistein stabilizes itself near the substrate/product binding site by moving into an extended binding site (Figure 3 in S1 Appendix). This indicates that behavior of genistein at the cPEPCK binding is different in the presence of GTP than that in unbound cPEPCK. In the presence of GTP, genistein stabilizes itself at the substrate/product binding site as opposed to moving towards GTP binding site in absence of any cofactor at cPEPCK binding site. Binding free energy (ΔG) of genistein with GTP_cPEPCK was estimated to be -23.26/-16.75 kcal/mol(±3.42) using MMGB/PBSA method (Table 1).

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