Abstract
Most investigations of the allosteric properties of the regulatory enzyme aspartate transcarbamoylase (ATCase) from Escherichia coli are based on the sigmoidal dependence of enzyme activity on substrate concentration and the effects of the inhibitor, CTP, and the activator, ATP, on the saturation curves. Interpretations of these effects in terms of molecular models are complicated by the inability to distinguish between changes in substrate binding and catalytic turnover accompanying the allosteric transition. In an effort to eliminate this ambiguity, the binding of the 3H-labeled bisubstrate analog N-(phosphonacetyl)-L-aspartate (PALA) to aspartate transcarbamoylase in the absence and presence of the allosteric effectors ATP and CTP has been measured directly by equilibrium dialysis at pH 7 in phosphate buffer. PALA binds with marked cooperativity to the holoenzyme with an average dissociation constant of 110 nM. ATP and CTP alter both the average affinity of ATCase for PALA and the degree of cooperativity in the binding process in a manner analogous to their effects on the kinetic properties of the enzyme; the average dissociation constant of PALA decreases to 65 nM in the presence of ATP and increases to 266 nM in the presence of CTP while the Hill coefficient, which is 1.95 in the absence of effectors, becomes 1.35 and 2.27 in the presence of ATP and CTP, respectively. The isolated catalytic subunit of ATCase, which lacks the cooperative kinetic properties of the holoenzyme, exhibits only a very slight degree of cooperativity in binding PALA. The dissociation constant of PALA from the catalytic subunit is 95 nM. Interpretation of these results in terms of a thermodynamic scheme linking PALA binding to the assembly of ATCase from catalytic and regulatory subunits demonstrates that saturation of the enzyme with PALA shifts the equilibrium between holoenzyme and subunits slightly toward dissociation. Ligation of the regulatory subunits by either of the allosteric effectors leads to a change in the effect of PALA on the association-dissociation equilibrium.
Highlights
Cooperative Binding of theBisubstrate Analog N-(Phosphonacety1)-Laspartate to Aspartate Transcarbamoylase andthe Heterotropic Effects of ATP and CTP*
ATP and CTP altebroth of velocity on substrate concentration or the average affinityof ATCase for PALA and thede- sensitivity to a non-substrate ligand binding at a regulatory gree of cooperativity in the binding processin a man- site
Considerable uncerner analogous to their effecotsn the kinetic properties tainty attends the interpretation of steady state kinetics in of the enzyme; the average dissociationconstant of terms of ligand binding
Summary
Chemicals-~-I2,3-~H]Aspartaiccid (14.9 Ci/mmol) was obtained from Du Pont-New England Nuclear. The procedure was modified slightly to include readjustment of the reaction mixture to approximately pH 8 by the addition of 5 M NaOH after each addition of the acid chloride This modification led to a substantial increase in the yield of 13H] PALA. The concentration of free ligand at halfsolved and addition of C subunit tothe solution was repeated, saturation of the enzyme, andthe average dissociation the amount of enzyme was sufficient to provide only a 20% excess of active sites over [3H]PALA.The solution was treated as described above with several cycles of concentration and buffer addition, and theextraction procedure was repeated. In the presence of ATP, K0.5 and K,, decreased to 53 nM and 65 nM, respectively, and increased to waslyophilized and stored at -20 “C until use This purification procedure was designed to allow the removal of any reaction compo-. Since phosphate bindsto the assayed for radiochemical purity by chromatography on polyethyl- active site of the enzyme, the apparent dissociation constant detereneimine-impregnated cellulose
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