Interactive binding between the substrate and allosteric sites of carbamoyl-phosphate synthetase.
The interaction between Escherichia coli carbamoyl-phosphate synthetase (CPS) and a fluorescent analogue of an allosteric effector molecule, 1,N6-ethenoadenosine 5'-monophosphate (epsilon-AMP), has been detected by using fluorescence techniques and kinetic measurements. From fluorescence anisotropy titrations, it was found that epsilon-AMP binds to a single site on CPS with Kd = 0.033 mM. The nucleotide had a small activating effect on the rate of synthesis of carbamoyl phosphate but had no effect on the Km for ATP. To test whether epsilon-AMP binds to an allosteric site, allosteric effectors (UMP, IMP, and CMP), known to bind at the UMP/IMP site, were added to solutions containing the epsilon-AMP-CPS complex. With addition of these effector molecules, a progressive decrease of the fluorescence anisotropy was observed, indicating that bound epsilon-AMP was displaced by the allosteric effectors examined. From these titrations, the dissociation constants for UMP, IMP, CMP, ribose 5-phosphate, 2-deoxyribose 5-phosphate, and orthophosphate were determined. When MgATP, a substrate, was employed as a titrant, the observed decrease in anisotropy was consistent with the formation of a ternary complex (epsilon-AMP-CPS-MgATP). The effect of ATP binding, monitored at the allosteric site, was magnesium dependent, and free magnesium in solution was required to obtain a hyperbolic binding isotherm. Solvent accessibility of epsilon-AMP in binary (epsilon-AMP-CPS) and ternary (epsilon-AMP-CPS-MgATP) complexes was determined from acrylamide quenching, showing that the base of epsilon-AMP is well shielded from the solvent even in the presence of MgATP.(ABSTRACT TRUNCATED AT 250 WORDS)
Kasprzak
A A
AA
Department of Chemistry, Pennsylvania State University, University Park 16802.
Villafranca
J J
JJ
D000249:Adenosine Monophosphate / Q000031:analogs & derivatives / Q000378:metabolism; D000495:Allosteric Site; D001665:Binding Sites; D002222:Carbamoyl-Phosphate Synthase (Ammonia) / Q000378:metabolism*; D004926:Escherichia coli / Q000201:enzymology; D005454:Fluorescence Polarization; D007700:Kinetics; D008433:Mathematics; D008954:Models, Biological
C030368:etheno-AMP; D000249:Adenosine Monophosphate; D002222:Carbamoyl-Phosphate Synthase (Ammonia)