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.
Yoon, Moon Young; Thayer-Cook, Kim A.; Berdis, Anthony J.; Karsten, William E.; Schnackerz, Klaus D.; and Cook, Paul F., "Acid-Base Chemical Mechanism of Aspartase From Hafnia alvei" (1995). Chemistry Faculty Publications. 168.
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