Mechanism-based inactivation of L-aspartase from Escherichia coli.
The substrate analogue L-aspartate beta-semialdehyde (L-ASA) has been identified as a mechanism-based inactivator of L-aspartase from Escherichia coli. The enzyme catalyzes the deamination of L-ASA to yield fumaric acid semialdehyde (FAA) and NH4+, with the product FAA partitioning between subsequent release or irreversible enzyme inactivation. Complete protection against L-ASA inactivation is observed in the presence of the product fumarate and a divalent metal ion. However, protection against inactivation by the product FAA also requires the presence of an enzyme activator. In addition to functioning as a mechanism-based inactivator, L-ASA has also been shown to serve as an activator of L-aspartase. The mechanism of inactivation by FAA involves the attack of an active site nucleophilic at the alpha-carbon of FAA to yield a stable Michael type enzyme adduct. Subsequent formation of a hydrazone upon treatment of the enzyme adduct with 2,4-dinitrophenylhydrazine confirms the presence of the unreacted aldehydic group of FAA. Examination of a group of product analogues with different substituents has demonstrated a correlation between the electron-withdrawing ability of these functional groups and the rate of inactivation of L-aspartase.
Schindler
J F
JF
Department of Chemistry, University of Akron, Ohio 44325-3601.
Viola
R E
RE
D001220:Aspartate Ammonia-Lyase / Q000037:antagonists & inhibitors* / Q000378:metabolism; D001224:Aspartic Acid / Q000031:analogs & derivatives* / Q000378:metabolism / Q000494:pharmacology; D004789:Enzyme Activation; D004926:Escherichia coli / Q000201:enzymology*; D006863:Hydrogen-Ion Concentration; D007700:Kinetics; D008433:Mathematics; D008962:Models, Theoretical; D010659:Phenylhydrazines / Q000494:pharmacology
D010659:Phenylhydrazines; C004787:2,4-dinitrophenylhydrazine; D001224:Aspartic Acid; C006644:aspartic semialdehyde; D001220:Aspartate Ammonia-Lyase