BIOL 268 |
| The energetic contributions of active site side chains to enzyme activity can be complex and difficult to recreate in computational protein design calculations. To evaluate the sequence-space landscape for stability and catalysis in the E. coli monofunctional P-protein chorismate mutase, we used site-saturation mutagenesis to place all 20 amino acids in six non-critical active site positions. The ability of each mutant to promote viability in a chorismate mutase deletion strain was assessed, and active variants were purified and evaluated with respect to in vitro enzyme activity, far-UV circular dichroism, and thermal denaturation profiles. The results were compared to computational energies from enzyme design calculations. In some positions, complex factors remain elusive to a molecular mechanics and rotamer library-based approach. In other positions, however, enzyme design calculations are able to predict mutations that actually increase activity or stability relative to the wild-type enzyme. |
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Enzymes
4:30 PM-6:30 PM, Wednesday, 13 September 2006 Moscone Center -- Hall D, Poster
Sci-Mix
Division of Biological Chemistry |