| STUDY |
THEORY CAN ALSO be used to zoom in on the crux of a protein's behavior with unprecedented precision. Virginia W. Cornish, assistant chemistry professor at Columbia University, and her colleagues are applying this strategy to help understand the evolution of the bacterial enzyme responsible for penicillin resistance. The Achilles' heel of penicillin-sensitive bacteria is the penicillin binding protein (PBP), an enzyme that's essential for building cell walls but which is inactivated when it encounters the antibiotic. Penicillin-resistant bacteria, however, carry an additional enzyme, -lactamase, which instead hydrolyzes the antibiotic, rendering it powerless. -Lactamase likely evolved from an ancient PBP, but what exactly happened to the enzyme has remained a mystery. The proteins are remarkably alike. They have similar three-dimensional structures and conserved active-site residues. Yet their penicillin-hydrolyzing rate constants differ by about six orders of magnitude. "Our hope is to begin to understand what's responsible for the difference in chemical reactivity," Cornish said. -------------------------------------------------------------------------------- EVOLUTION A penicillin-binding protein, showing residues where mutations occurred (pink). Courtesy Of Shalom Goldberg -------------------------------------------------------------------------------- TO THAT END, Cornish and graduate student Shalom Goldberg are trying to "evolve" a PBP into a -lactamase. They've collaborated with Columbia chemistry professor Richard A. Friesner and his graduate student Benjamin F. Gherman in a computational study of the proteins. They combined quantum mechanics and classical molecular mechanics, treating the bulk of the protein as a classical blob of noncovalent interactions, while saving the more detailed and intensive quantum mechanical calculations for the few amino acids in the protein's active site. The researchers modeled both the ground and transition state of the hydrolysis reaction for both PBP and the -lactamase. Their calculations pointed to a single tyrosine residue, which is stabilized by a hydrogen-bonding network in the b-lactamase, allowing it to act as a general base catalyst. The residue isn't stabilized in PBP, however. PBP's active site looks like that of the -lactamase, "just slightly more crowded," Cornish said. Now the team is beginning to use this information to guide mutagenesis experiments. In directed evolution experiments, they've been able to increase the activity of PBP by an order of magnitude. "I think this is a trend in the field--to marry strengths in computation with the strengths of directed evolution to solve problems we haven't been able to solve yet," Cornish said |
| UPDATE | 10.03 |
| AUTHOR |
Columbia University's - Cornish Virginia W. & - Friesner Richard A. |
| LITERATURE REF. | This data is not available for free |
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