Interaction of Aspartate Transcarbamylase with Regulatory Nucleotides

Carla Winlund Gray,Michael J Chamberlin

doi:10.1016/s0021-9258(19)43510-2

Carla Winlund Gray, Michael J Chamberlin

Open Access

https://doi.org/10.1016/s0021-9258(19)43510-2

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Abstract

Abstract Equilibrium dialysis measurements of the binding of the allosteric activator ATP and the allosteric inhibitor CTP to native aspartate transcarbamylase show that ATP is bound at 5.1 to 6 sites with similar affinities, whereas Scatchard plots of CTP binding data display a sharp negative curvature indicating that CTP is bound with markedly heterogeneous affinities. ATP binds more weakly than does CTP, and the binding affinities of both nucleotides are decreased several fold by an increase in temperature from 4° to 24°. The binding affinity of CTP is reduced about 2-fold when the catalytic sites of aspartate transcarbamylase are occupied by a tightly bound transition-state analogue, N-(phosphonacetyl)-l-aspartate (PALA); there is no measurable effect of PALA on the binding of ATP. The heterogeneity in CTP binding affinities is observed under all conditions tested, in a manner consistent with the suggestion that heterogeneity in CTP binding may be due to nonequivalent binding of CTP to sites on dimers of regulatory polypeptide chains in the enzyme. Measurements have been made of the 190 to 320 nm circular dichroism (CD) spectrum of unliganded native aspartate transcarbamylase and of the CD difference spectra arising due to interaction of the enzyme with the substrate analogue PALA, the activator ATP, or the inhibitor CTP, taking these ligands singly or in combination. An estimate of polypeptide configurations in native aspartate transcarbamylase is presented, based on analysis of the 200 to 240 nm CD. Temperature-dependent spectral changes are not detected in the unliganded enzyme but are found in the difference CD at 250 to 320 nm for interaction of ATP or CTP with aspartate transcarbamylase. PALA and CTP added simultaneously to aspartate transcarbamylase have effects on the 200 to 250 nm CD which are not equal to the sum of the effects of PALA or CTP acting separately, reflecting the interdependence in the interaction of these two ligands with aspartate transcarbamylase. PALA, which is an analogue combining features of both of the substrates of aspartate transcarbamylase, produces changes in the 250 to 320 nm CD which are qualitatively and quantitatively similar to the sum of the changes observed by other workers for interaction of aspartate transcarbamylase with one substrate and an analogue of the other substrate.

Highlights

5.1 to 6 sites with similar affinities, whereas Scatchard plots of CTP binding data display a sharp “negative” curvature indicating that CTP is bound with markedly heterogeneous affinities
Temperature-dependent spectral changes are not detected in the unliganded enzyme but are found in the difference circular dichroism (CD) at 250 to 320 nm for interaction of ATP or CTP with aspartate
We find that 200 to 320 nm CD spectra taken at 4”, 8’, and 24” are essentially identical; we can detect no temperature-dependent changes in protein structure or conformation which give rise to an altered protein CD

Summary

SUMMARY

5.1 to 6 sites with similar affinities, whereas Scatchard plots of CTP binding data display a sharp “negative” curvature indicating that CTP is bound with markedly heterogeneous affinities. We are interested in characterizing the nucleotide binding sites of native aspartate transcarbamylasc with regard to their interaction with an activator, ATP, and au inhibitor, CTP, and in determining the effect of a substrate analogue on interactions of the regulatory binding sites with these nucleotide effecters. Other direct measurements of the binding of CTP to native aspartate transcarbamylase [4, 6, 17] are quantitatively consistent with these observations and &h those embodied in the present report, lvith the exception that the ultracentrifugal methods [4, 17] have tended to indicate a larger number of “low affinity”. III this rcxport we comparc~ the binding of ATP and CTI’ to native as1jartat.e trailscarljarn~lase undo identical conditions, in each (‘ax measuring the cfiect of variccl temperature and of a tightly bound substrate ailalogur, N-(phosphonacetyl)-L-aspartate (1’GA). Thosc~ results arc discussed in terms of a model for the intcractiotl ol’ thrst: nucleotitlcs with the regulatory hinfling sites 011 aspaitatc: tralisf:arbamylasc:

Our measurements

ATP BINDING ii

Circular Dichroism Xeasurements

Findings

CD Difference

DISCUSSION