HETEROTROPIC EFFECTORS PROMOTE A GLOBAL CONFORMATIONAL CHANGE IN ASPARTATE TRANSCARBAMOYLASE

The sigmoidal dependence of activity on substrate concentration exhibited by the regulatory enzyme aspartate transcarbamoylase (ATCase) of Escherichia coli is generally attributed to a ligand-promoted change in the quaternary structure of the enzyme. Although a global conformational change in ATCase upon the binding of ligands to some of the six active sites is well documented, a corresponding alteration in the structure of the wild-type enzyme upon the addition of the inhibitor, CTP, or the activator, ATP, has not been detected. Such evidence is essential for testing whether heterotropic, as well as homotropic, effects can be accounted for quantitatively in terms of coupled equilibria involving a conformational change in the enzyme and preferential binding of ligands to one conformation or the other. This evidence has now been obtained with a mutant form of ATCase in which Lys 143 in the regulatory chain was replaced by Ala, thereby perturbing interactions at the interface between the regulatory and catalytic chains in the enzyme and destabilizing the low-activity, compact (T) conformation relative to the high-activity, swollen (R) state. Difference sedimentation velocity experiments involving measurements of the changes caused by the binding of the bisubstrate analogue N-(phosphonacetyl)-l-aspartate demonstrated that the sedimentation coefficient of the mutant enzyme was intermediate between that observed for the T and R states of wild-type ATCase. We interpret the results as indicating that the [T]/[R] ratio in phosphate buffer at pH 7.0 is reduced from about 2 x 102 for the wild-type enzyme to 2.7 for r 143Ala ATCase. The addition of CTP to the mutant enzyme led to an increase in [T]/[R] to 34, whereas the ratio was lowered to 0.2 in the presence of ATP. Equilibrium dialysis measurements for the binding of PALA demonstrated that the Hill coefficient, nH, which was 1.4 in the absence of effectors, was increased to 2.0 in the presence of CTP and decreased to 1.0 by the addition of ATP. Analysis of the equilibrium binding data, in terms of the two-state model of Monod, Wyman, and Changeux, yielded [T]/[R] ratios in accord with those deduced from the sedimentation velocity experiments. These results provide convincing evidence that heterotropic effectors cause an alteration in the average quaternary structure of ATCase and thereby regulate the activity of the enzyme by perturbing the T ⇌ R equilibrium. Moreover, the observations refute suggestions that only local changes in the structure of the enzyme occur upon the binding of CTP and ATP. © 1990, American Chemical Society. All rights reserved.