Group:SMART:2010 Pingry SMART Team: Difference between revisions

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'''Design description'''
'''Design description'''
Four mutations of 2,5-Diketo-d-gluconic acid reductase have been conducted to alternate its cofcator specificity to <scene name='2010_Pingry_SMART_Team/1m9h_original/16'>NADH (shown in wireframe and colored CPK)</scene> rather than NADPH. These mutations of <scene name='2010_Pingry_SMART_Team/1m9h_original/17'>(Lys232Gly, Phe22Tyr, Arg238His, Ala272Gly).</scene>and their backbones have been highlighted orange to distinguish the change in amino acids between the 2,5-DKGR wildtype and the NADP-binding mutant.


Mutations of the 2,5-DKGR's conformation have been conducted for alternate cofactor specificity to <scene name='2010_Pingry_SMART_Team/1m9h_original/16'>NADH (shown in wireframe and colored CPK)</scene> rather than NADPH.


The backbone of the four residues changed between WT and NADP-binding mutant are colored orange <scene name='2010_Pingry_SMART_Team/1m9h_original/17'>(Lys232Gly, Phe22Tyr, Arg238His, Ala272Gly).</scene>. The <scene name='2010_Pingry_SMART_Team/1m9h_original/2'>Lys232Gly mutation
Lys 232 in the 2,5-DKGR wildtype interacts directly with the pyrophosphate group of NADPH through hydrogen bonds. However, in the 2,5-DKGR mutant,this residue has been altered into a<scene name='2010_Pingry_SMART_Team/1m9h_original/2'>Lys232Gly mutation</scene>
</scene> is important because Gly has no side chain and there is no need for the Lys side chain to interact with a phosphate group because the phosphate group is absent in this mutation. The <scene name='2010_Pingry_SMART_Team/1m9h_original/3'>Phe22Tyr mutation</scene> reduces the Km for both NADPH and NADH. A reduced Km makes a more efficient enzyme at a lower substrate level, therefore, improving the enzyme. The Arg238His mutation forms a pi-stacking interaction to stabilize the AKR with the cofactor. A pi-stacking interaction is extremely stable and the pi bonds are perpendicular. A common source for pi-stacking is in DNA. The <scene name='2010_Pingry_SMART_Team/1m9h_original/5'>Ala272Gly mutation</scene> mutation improves the kinetic properties by making it easier for the substrate to bind with the substrate and by improving the kinetics of cofactor binding and release.
to adapt to the absent pyrophosphate group in NADH. Significantly, Gly lacks a side chain since no interaction is necessary due to the absent phosphate group in NADH. In addition, the mutation results with with a reduction in cofactor Ka and kcat.  
 
The <scene name='2010_Pingry_SMART_Team/1m9h_original/3'>Phe22Tyr mutation</scene> reduces the Km for both NADPH and NADH. A reduced Km makes a more efficient enzyme at a lower substrate level, therefore, improving the enzyme. The Arg238His mutation forms a pi-stacking interaction to stabilize the AKR with the cofactor. A pi-stacking interaction is extremely stable and the pi bonds are perpendicular. A common source for pi-stacking is in DNA. The <scene name='2010_Pingry_SMART_Team/1m9h_original/5'>Ala272Gly mutation</scene> mutation improves the kinetic properties by making it easier for the substrate to bind with the substrate and by improving the kinetics of cofactor binding and release.


The residue <scene name='2010_Pingry_SMART_Team/1m9h_original/6'>Trp187</scene> is highlighted by displaying the side chain. The pi-stacking interaction Trp187 has with the nicotinamide ring of the cofactor stabilizes the reaction.
The residue <scene name='2010_Pingry_SMART_Team/1m9h_original/6'>Trp187</scene> is highlighted by displaying the side chain. The pi-stacking interaction Trp187 has with the nicotinamide ring of the cofactor stabilizes the reaction.
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