13C isotope effect studies of Escherichia coli aspartate transcarbamylase in the presence of the bisubstrate analog N-(phosphonoacetyl)-L-aspartate.
13C isotope effects have been measured for the aspartate transcarbamylase holoenzyme (ATCase) and catalytic subunit catalyzed reactions in the presence of the bisubstrate analog N-(phosphonoacetyl)-L-aspartate (PALA). For holoenzyme-catalyzed reactions in the physiological direction with very low levels of L-aspartate as substrate, or with L-cysteine sulfinate as substrate, or in the reverse direction with carbamyl-L-aspartate and phosphate as substrates, the isotope effect data show a slight dependence on PALA concentration. Under these conditions, PALA first stimulates the rate and then inhibits it at higher concentrations. The observed isotope effect at maximum stimulation by PALA is slightly smaller than in the absence of the analog, but as the PALA concentration is increased to reduce the rate to its original value, the observed isotope effect also increases and approaches the value of the isotope effect determined in the absence of PALA. These data suggest that the kinetic properties of the active enzyme are affected by the number of active sites occupied by PALA, indicating communication between subunits, and a mathematical model is proposed which explains our experimental observations. In contrast to these results with the holoenzyme, isotope effects measured for the reaction catalyzed by the isolated catalytic subunits are not altered in the presence of PALA. Taken together, these data are consistent with the two-state model for the homotropic regulation of ATCase.
Parmentier
L E
LE
Institute for Enzyme Research, University of Wisconsin, Madison 53705.
O'Leary
M H
MH
Schachman
H K
HK
Cleland
W W
WW
D000494:Allosteric Regulation; D001221:Aspartate Carbamoyltransferase / Q000037:antagonists & inhibitors / Q000378:metabolism*; D001224:Aspartic Acid / Q000031:analogs & derivatives* / Q000378:metabolism; D002247:Carbon Isotopes; D004926:Escherichia coli / Q000201:enzymology; D006863:Hydrogen-Ion Concentration; D007700:Kinetics; D010746:Phosphonoacetic Acid / Q000031:analogs & derivatives* / Q000378:metabolism
D002247:Carbon Isotopes; D001224:Aspartic Acid; C013195:sparfosic acid; D001221:Aspartate Carbamoyltransferase; D010746:Phosphonoacetic Acid