BIOL 179 |
| To appreciate what has been accomplished during the evolution of modern enzymes, it is of interest to consider how closely the value of kcat/Km approaches the limits imposed by diffusional encounter. But it is also of interest to consider the inherent difficulty of the reaction that each enzyme catalyzes. Measurements at high temperatures allow the rate constants of uncatalyzed reactions to be evaluated at ambient temperatures, and furnish additional information about the thermodynamics of activation. Two contrasting examples will be discussed. 1) As a model for the hydrolytic reaction catalyzed by staphylococcal nuclease (water attack at the phosphorus atom of a phosphodiester), we examined the hydrolysis of dineopentyl phosphate (nP2P), in which water attack at carbon is sterically precluded. Measurements at elevated temperature indicate that the anion of nP2P is hydrolyzed with a half-time of 31,000,000 years at room temperature, so that staphylococcal nuclease produces one of the largest rate enhancements (1.4e17-fold) that is produced by any enzyme. That rate enhancement arises entirely from a lowering the enthalpy of activation. 2) As a model for peptidyl transfer from tRNA to the amino group of aminoacyl-tRNA in the ribosome, a two-substrate reaction in protein biosynthesis, we examined the reaction of the trifluoroethyl ester of formylglycine with glycinamide. When the second order rate constant for this model reaction was compared with the second order rate constant observed for ribosomal peptidyl transfer from tRNA to puromycin, the ribosome was found to enhance the rate of reaction by a factor of 2.3e6. That rate enhancement was found to be entirely entropic in origin. |
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Repligen Award Symposium: Enzymatic Catalysis and Transition States
1:30 PM-4:40 PM, Wednesday, 13 September 2006 Moscone Center -- Room 238, Oral
Division of Biological Chemistry |