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Archives of Biochemistry and Biophysics


An acid-base chemical mechanism is proposed for Hafnia alvei aspartase in which a proton is abstracted from C-3 of the monoanionic form of L-aspartate by an enzyme general base with a pK of 6.3-6.6 in the absence and presence of Mg2+. The resulting carbanion is presumably stabilized by delocalization of electrons into the β-carboxyl with the assistance of a protonated enzyme group in the vicinity of the β-carboxyl. Ammonia is then expelled with the assistance of a general acid group that traps an initially expelled NH3 as the final NH+4 product. In agreement with the function of the general acid group, potassium, an analog of NH+4, binds optimally when the group is unprotonated. The pK for the general acid is about 7 in the absence of Mg2+, but is increased by about a pH unit in the presence of Mg2+. Since the same pK values are observed in the pKisuccinate and V/K pH profile, both enzyme groups must be in their optimum protonation state for efficient binding of reactant in the presence of Mg2+. At the end of a catalytic cycle, both the general base and general acid groups are in a protonation state opposite that in which they started when aspartate was bound. The presence of Mg2+ causes a pH-dependent activation of aspartase exhibited as a partial change in the V and V/Kasp pH profiles. When the aspartase reaction is run in D2O to greater than 50% completion no deuterium is found in the remaining aspartate, indicating that the site is inaccessible to solvent during the catalytic cycle.


This work was supported by grants to P.F.C. from the National Institutes of Health (GM 36799) and the Robert A. Welch Foundation (B-1031) and to K.D.S. from the Deutsche Forschungsgemeinschaft (Schn 139/11-2).

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