Methotrexate: Difference between revisions
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The features of DHFR ligand binding, specifically to methotrexate can be observed and analyzed through various molecular docking and mutation experiments. The a structurally engineered variant of the <scene name='Sandbox_58/Arg_31-35_norm/1'>native human DHFR</scene> altered the F 31 residue of the protein to R, and the Q 35 residue of the protein to E in an attempt to explore the specifics of the methotrexate affinity for DHFR active site residues, resulting in varied active site residues from phenylalanine and glutamine to <scene name='Sandbox_58/Arg_31_glutamine_35/1'>arginine and glutamate</scene>. This mutated enzyme featured a 650x decrease in affinity for the ligand, methotrexate, but retained an amount of methotrexate interaction similar to the enzyme in its native state with native substrates. Crystal structure analysis revealed that the lack of cooperative action and presence of residue disorder lead to the significant decrease in methotrexate activity with the resulting <scene name='Sandbox_58/3eig_active_site/1'>active site</scene>. The arginine residue at place 31, was specifically observed in numerous conformations, a characteristic unique to the mutated enzyme, and the probable cause of the loss of polar contacts and binding affinity between methotrexate and DHFR. A loss of van der Waal forces due to the conformations of the side chains along with an unfavorable placement of Glu-35 causing an “unfavorable electrostatic contact” with methotrexate’s “glutamate portion.” Interestingly this variant was found to display a greater decrease in methotrexate affinity than the decrease in affinity of Dihydrofolate, found to be 9x, evident of catalytic efficiency retention which hold many drug binding resistance implications<ref>Volpato, J., Yachnin, B., & Blanchet, J. (2009). Multiple conformers in active site of human dihydrofolate reductase F31R/Q35E double mutant suggest structural basis for methotrexate resistance.. Journal Biol. Chem., 284, 20079-20089. </ref>. | The features of DHFR ligand binding, specifically to methotrexate can be observed and analyzed through various molecular docking and mutation experiments. The a structurally engineered variant of the <scene name='Sandbox_58/Arg_31-35_norm/1'>native human DHFR</scene> altered the F 31 residue of the protein to R, and the Q 35 residue of the protein to E in an attempt to explore the specifics of the methotrexate affinity for DHFR active site residues, resulting in varied active site residues from phenylalanine and glutamine to <scene name='Sandbox_58/Arg_31_glutamine_35/1'>arginine and glutamate</scene>. This mutated enzyme featured a 650x decrease in affinity for the ligand, methotrexate, but retained an amount of methotrexate interaction similar to the enzyme in its native state with native substrates. Crystal structure analysis revealed that the lack of cooperative action and presence of residue disorder lead to the significant decrease in methotrexate activity with the resulting <scene name='Sandbox_58/3eig_active_site/1'>active site</scene>. The arginine residue at place 31, was specifically observed in numerous conformations, a characteristic unique to the mutated enzyme, and the probable cause of the loss of polar contacts and binding affinity between methotrexate and DHFR. A loss of van der Waal forces due to the conformations of the side chains along with an unfavorable placement of Glu-35 causing an “unfavorable electrostatic contact” with methotrexate’s “glutamate portion.” Interestingly this variant was found to display a greater decrease in methotrexate affinity than the decrease in affinity of Dihydrofolate, found to be 9x, evident of catalytic efficiency retention which hold many drug binding resistance implications<ref>Volpato, J., Yachnin, B., & Blanchet, J. (2009). Multiple conformers in active site of human dihydrofolate reductase F31R/Q35E double mutant suggest structural basis for methotrexate resistance.. Journal Biol. Chem., 284, 20079-20089. </ref>. | ||
[[Image:2011-03-10 2241.png| | [[Image:2011-03-10 2241.png|400px|left|thumb| Methotrexate Variant Residue Interaction <ref>DIHYDROFOLATE REDUCTASE COMPLEXED WITH METHOTREXATE. (n.d.). RCSB Protein Database. Retrieved March 10, 2011, from www.rcsb.org/pdb/results </ref>]] | ||
== Pharmaceutical Implications == | == Pharmaceutical Implications == | ||
