Acid-Base Chemical Mechanism of Aspartase from Hafnia alvei

• Published in: Archives of biochemistry and biophysics 1995-06, Vol.320 (1), p.115-122
• Main Authors: Yoon, M.Y., Thayercook, K.A., Berdis, A.J., Karsten, W.E., Schnackerz, K.D., Cook, P.F.
• Format: Article
• Language: English
• Subjects: Acid-Base Equilibrium; Aspartate Ammonia-Lyase - chemistry; aspartate-ammonia ligase; Aspartic Acid - metabolism; deuterium oxide; Enterobacteriaceae - enzymology; enzyme activity; enzyme reaction mechanisms; Escherichia coli - enzymology; Fumarates - metabolism; Hafnia alvei; Hydrogen-Ion Concentration; isotopes; Kinetics; magnesium; Magnesium - pharmacology; Models, Chemical; Succinates - metabolism; Succinic Acid
• ISSN: 0003-9861; 1096-0384
• DOI: 10.1006/abbi.1995.1348

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.