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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).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.

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

Binding of genistein with u_cPEPCK.A) Superimposed structure of u_cPEPCK-genistein complex and OAA-GTP_cPEPCK complex to identify location of genistein at binding site of u_cPEPCK after docking. Genistein (blue) can clearly be seen located at the site of GTP (red) binding. B) 2D representation of interaction of genistein with u_cPEPCK protein.
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pone.0141987.g003: Binding of genistein with u_cPEPCK.A) Superimposed structure of u_cPEPCK-genistein complex and OAA-GTP_cPEPCK complex to identify location of genistein at binding site of u_cPEPCK after docking. Genistein (blue) can clearly be seen located at the site of GTP (red) binding. B) 2D representation of interaction of genistein with u_cPEPCK protein.

Mentions: Absence of GTP or GDP at binding site in u_cPEPCK offers a larger cavity for ligand binding. Within large cavity only strong intermolecular interactions can hold a ligand stably. Hence, to assess the genistein binding with u_cPEPCK, prepared structure of genistein was docked with refined u_cPEPCK structure. Autodock score of -6.52 kcal/mol was observed indicating a strong affinity between the two molecules (Table 1). Semi-flexible docking methods do not incorporate the changes that take place in the protein structure due to ligand. Hence, to take into consideration the effect of genistein binding on the u_cPEPCK binding site, the docked structure was further refined by a ligand refinement web server PELE. Using the OPLS force field, the ligand binding refinement algorithm performed translations combined with small and large rotations of genistein within the binding site. Refinement generated the best docking pose of genistein within the binding site on u_cPEPCK, with RMSD of 6.62 Å in reference to the initial position of the ligand. The PELE binding score of -37.77 was obtained. When binding position of genistein was compared with GTP bound conformation of cPEPCK, it was found that genistein preferentially binds at GTP binding site in the absence of any co-factor at the binding site (Fig 3A). Genistein formed hydrogen bonds with Lys244, His264, Thr291, Tyr235, and Thr339 while hydrophobic interactions were seen with residues Ser286, Lys290 and Val335 (Table 2 and S3A Fig). Most of these residues were present within 4Å vicinity of genistein (Fig 3B). Ala287, Thr291, Lys290, and Val235 of u_cPEPCK involved in interaction with genistein were the residues that in general help in holding GTP at its binding site. Hence, it can be concluded that genistein in the absence of any cofactor preferentially binds to the GTP binding site of the enzyme.


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)

Binding of genistein with u_cPEPCK.A) Superimposed structure of u_cPEPCK-genistein complex and OAA-GTP_cPEPCK complex to identify location of genistein at binding site of u_cPEPCK after docking. Genistein (blue) can clearly be seen located at the site of GTP (red) binding. B) 2D representation of interaction of genistein with u_cPEPCK protein.
© Copyright Policy
Related In: Results  -  Collection

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

pone.0141987.g003: Binding of genistein with u_cPEPCK.A) Superimposed structure of u_cPEPCK-genistein complex and OAA-GTP_cPEPCK complex to identify location of genistein at binding site of u_cPEPCK after docking. Genistein (blue) can clearly be seen located at the site of GTP (red) binding. B) 2D representation of interaction of genistein with u_cPEPCK protein.
Mentions: Absence of GTP or GDP at binding site in u_cPEPCK offers a larger cavity for ligand binding. Within large cavity only strong intermolecular interactions can hold a ligand stably. Hence, to assess the genistein binding with u_cPEPCK, prepared structure of genistein was docked with refined u_cPEPCK structure. Autodock score of -6.52 kcal/mol was observed indicating a strong affinity between the two molecules (Table 1). Semi-flexible docking methods do not incorporate the changes that take place in the protein structure due to ligand. Hence, to take into consideration the effect of genistein binding on the u_cPEPCK binding site, the docked structure was further refined by a ligand refinement web server PELE. Using the OPLS force field, the ligand binding refinement algorithm performed translations combined with small and large rotations of genistein within the binding site. Refinement generated the best docking pose of genistein within the binding site on u_cPEPCK, with RMSD of 6.62 Å in reference to the initial position of the ligand. The PELE binding score of -37.77 was obtained. When binding position of genistein was compared with GTP bound conformation of cPEPCK, it was found that genistein preferentially binds at GTP binding site in the absence of any co-factor at the binding site (Fig 3A). Genistein formed hydrogen bonds with Lys244, His264, Thr291, Tyr235, and Thr339 while hydrophobic interactions were seen with residues Ser286, Lys290 and Val335 (Table 2 and S3A Fig). Most of these residues were present within 4Å vicinity of genistein (Fig 3B). Ala287, Thr291, Lys290, and Val235 of u_cPEPCK involved in interaction with genistein were the residues that in general help in holding GTP at its binding site. Hence, it can be concluded that genistein in the absence of any cofactor preferentially binds to the GTP binding site of the enzyme.

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).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.

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