ACID-BASE CHEMICAL MECHANISM OF ASPARTASE FROM HAFNIA-ALVEI

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 absen ce and presence of Mg2+, The resulting carbanion is presumably stabili zed by delocalization of electrons into the beta-carboxyl with the ass istance of a protonated enzyme group in the vicinity of the beta-carbo xyl, Ammonia is then expelled with the assistance of a general acid gr oup that traps an initially expelled NH3 as the final NH4+ product, In agreement with the function of the general acid group, potassium, an analog of NH4+, binds optimally when the group is unprotonated, The pK for the general acid is about 7 in the absence of Mg2+, but is increa sed by about a pH unit in the presence of Mg2+, Since the same pK valu es are observed in the pK(i) (succinate) and V/K pH profile, both enzy me groups must be in their optimum protonation state for efficient bin ding of reactant in the presence of Mg2+, At the end of a catalytic cy cle, both the general base and general acid groups are in a protonatio n state opposite that in which they started when aspartate was bound, The presence of Mg2+ causes a pH-dependent activation of aspartase exh ibited as a partial change in the V and V/K-asp PH profiles. When the aspartase reaction is run in D2O to greater than 50% completion no deu terium is found in the remaining aspartate, indicating that the site i s inaccessible to solvent during the catalytic cycle. (C) 1995 Academi c Press, Inc.