13C and 15N isotope effects as a probe of the chemical mechanism of Escherichia coli aspartate transcarbamylase.
13C and 15N isotope effects have been measured for the aspartate transcarbamylase (ATCase) reaction in an effort to elucidate the chemical mechanism of this highly regulated enzyme. The observed 15(V/K(asp))H2O value for the ATCase holoenzyme at saturing carbamyl phosphate and 12 mM L-aspartate is 1.0045 at pH 7.5, and this value remains unchanged in the presence of 5 mM ATP (activator) or 5 mM CTP (inhibitor). The fact that the isotope effect is not changed by the allosteric modifiers supports the conclusion that the kinetic properties of the active form of ATCase are not influenced by ATP or CTP. The observed 15(V/K(asp)) values for the catalytic subunit of ATCase are also the same as those determined for the holoenzyme, suggesting that the chemical mechanisms of both enzyme species are the same. Quantitative analysis of 13C and 15N isotope effects in both H2O and D2O has led to the proposal of a chemical model for the ATCase reaction which involves a precatalytic conformational change to form an activated complex that facilitates deprotonation of L-aspartate by an enzyme functional group. Nucleophilic attack on the carbonyl carbon of carbamyl phosphate by the alpha-amino group of L-aspartate results in the formation of a tetrahedral intermediate. An intramolecular proton transfer leads to formation of products N-carbamyl-L-aspartate and inorganic phosphate.
Parmentier
L E
LE
Department of Chemistry, University of Wisconsin, Madison 53705.
Weiss
P M
PM
O'Leary
M H
MH
Schachman
H K
HK
Cleland
W W
WW
D001221:Aspartate Carbamoyltransferase / Q000378:metabolism*; D002247:Carbon Isotopes; D002384:Catalysis; D003903:Deuterium; D004926:Escherichia coli / Q000201:enzymology*; D066298:In Vitro Techniques; D007700:Kinetics; D046911:Macromolecular Substances; D009587:Nitrogen Isotopes
D002247:Carbon Isotopes; D046911:Macromolecular Substances; D009587:Nitrogen Isotopes; D003903:Deuterium; D001221:Aspartate Carbamoyltransferase