The replacement of Lys258 by alanine (K258A) in aspartate aminotransferase reduces the rate constant for the central, 1,3-prototropic shift by 106-108-fold, confirming the role of Lys258 as the generalbase catalyst for this step. The rate constant for the 1,3-prototropic shift interconverting K258A aldimine and ketimine intermediates is pH-independent, like that of the wild-type enzyme (WT-AATase). K258A binds amino acid substrates in external aldimine intermediates 105-fold more tightly than does WT-AATase. The excess amino acid binding energy observed in the mutant is sacrificed by the WT-AATase in order to increase the value of kcat. The net result is that the kcat/KM values for amino acid substrates are reduced only 3-100-fold by the mutation. This provides a clear example of the Circe effect propounded by Jencks [Jencks, W. P. (1975) Adv. Enzymol. Rel. Areas Mol. Biol. 43, 219]. Part of the increase in kcat due to the inclusion of Lys258 is accomplished by a 104-105-fold acceleration of external aldimine formation and hydrolysis. This step is partially rate-determining for K258A, but not for WT-AATase. A significant consequence of the utilization of amino acid binding energy for catalysis is the raising of the dissociation constants for these substrates to levels near the physiological concentrations of amino acids. The major product of the reaction of K258 A with oxalacetate is pyruvate due to decarboxylation of the β-imine formed in the ketimine intermediate.
|Original language||English (US)|
|Number of pages||9|
|State||Published - 1993|
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