13C isotope effects as a probe of the kinetic mechanism and allosteric properties of Escherichia coli aspartate transcarbamylase.
13C kinetic isotope effects have been measured in carbamyl phosphate for the reaction catalyzed by aspartate transcarbamylase. For the holoenzyme, the value was 1.0217 at zero aspartate, but unity at infinite aspartate, with 4.8 mM aspartate eliminating half of the isotope effect. This pattern proves an ordered kinetic mechanism, with carbamyl phosphate adding before aspartate. The same parameters were observed in the presence of ATP or CTP, showing that there is only one form of active enzyme present, regardless of the presence or absence of allosteric modifiers. These data support the Monod model of allosteric behavior in which the equilibrium between fully active and inactive enzyme is perturbed by selective binding interactions of substrates and modifiers, and there are no enzyme forms having partial activity. Isolated catalytic subunits of the enzyme showed similar 13C isotope effects (1.0240 at zero aspartate, 1.0039 at infinite aspartate, 3.8 mM aspartate causing half of the change from one value to the other), but the finite isotope effect at infinite aspartate shows that the kinetic mechanism is now partly random. With the very slow and poorly bound aspartate analog cysteine sulfinate, the 13C isotope effects were 1.039 for both holoenzyme and catalytic subunits and were not decreased significantly by high levels of cysteine sulfinate. The value of 1.039 is probably close to the intrinsic isotope effect on the chemical reaction, while the kinetic mechanism with this substrate is now fully random because the chemistry is so much slower than release of either reactant from the enzyme.
Parmentier
L E
LE
Department of Chemistry, University of Wisconsin, Madison 53705.
O'Leary
M H
MH
Schachman
H K
HK
Cleland
W W
WW
D000255:Adenosine Triphosphate / Q000378:metabolism; D000494:Allosteric Regulation; D001221:Aspartate Carbamoyltransferase / Q000378:metabolism*; D001224:Aspartic Acid / Q000378:metabolism; D002221:Carbamyl Phosphate / Q000378:metabolism; D002247:Carbon Isotopes; D003545:Cysteine / Q000031:analogs & derivatives / Q000378:metabolism; D003570:Cytidine Triphosphate / Q000378:metabolism; D004926:Escherichia coli / Q000201:enzymology; D006863:Hydrogen-Ion Concentration; D066298:In Vitro Techniques; D007700:Kinetics; D046911:Macromolecular Substances; D018377:Neurotransmitter Agents
D002247:Carbon Isotopes; D046911:Macromolecular Substances; D018377:Neurotransmitter Agents; D001224:Aspartic Acid; D002221:Carbamyl Phosphate; D003570:Cytidine Triphosphate; D000255:Adenosine Triphosphate; D001221:Aspartate Carbamoyltransferase; D003545:Cysteine; C013461:cysteine sulfinic acid