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 absence and presence of Mg2+. The resulting carbanion is presumably stabilized by delocalization of electrons into the beta-carboxyl with the assistance of a protonated enzyme group in the vicinity of the beta-carboxyl. Ammonia is then expelled with the assistance of a general acid group 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 increased by about a pH unit in the presence of Mg2+. Since the same pK values are observed in the pKi(succinate) 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
M Y
MY
Department of Biochemistry and Molecular Biology, University of North Texas Health Science Center, Fort Worth 76107, USA.
Thayer-Cook
K A
KA
Berdis
A J
AJ
Karsten
W E
WE
Schnackerz
K D
KD
Cook
P F
PF
D000136:Acid-Base Equilibrium; D001220:Aspartate Ammonia-Lyase / Q000737:chemistry*; D001224:Aspartic Acid / Q000378:metabolism; D004755:Enterobacteriaceae / Q000201:enzymology*; D004926:Escherichia coli / Q000201:enzymology; D005650:Fumarates / Q000378:metabolism; D006863:Hydrogen-Ion Concentration; D007700:Kinetics; D008274:Magnesium / Q000494:pharmacology; D008956:Models, Chemical; D013386:Succinates / Q000378:metabolism; D019802:Succinic Acid
D005650:Fumarates; D013386:Succinates; D001224:Aspartic Acid; C032005:fumaric acid; D019802:Succinic Acid; D001220:Aspartate Ammonia-Lyase; D008274:Magnesium