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Spectral alterations associated with the ligand promoted gross conformational change in aspartate trans carbamoylase ec

, : Spectral alterations associated with the ligand promoted gross conformational change in aspartate trans carbamoylase ec Journal of Biological Chemistry 256(10): 4998-5004

Spectral studies on aspartate transcarbamoylase [EC] of Escherichia coli showed that the difference spectra for binding each molar equivalent of the bisubstrate analog, N-(phosphonacetyl)-L-aspartate, varied according to the degree of saturation of the 6 active sites. In contrast, the progressive binding of the same ligand to the free catalytic subunit yielded difference spectra which were independent of the extent of occupancy of the 3 active sites. These observations in conjunction with results from other physicochemical studies of the gross conformational change in the enzyme are interpreted in terms of the sum of 2 spectral changes. One represents the local effect of ligand binding and the 2nd corresponds to the conversion of the enzyme from the constrained, low affinity, T-state to a relaxes conformation of higher affinity (R-state). This transition is virtually complete when only about 4 of the 6 active sites are occupied by the bisubstrate analog. Hence, subtraction of the difference spectrum for the binding of the last 2 ligand molecules from the spectrum for binding the first 2 molecules yielded the spectral change for the conversion of about 50% of the enzyme from the T- to the R-state. A more reliable measurement of the difference spectrum corresponding to the gross conformational change of unliganded enzyme was obtained by comparing the total spectral change (local and gross) for binding ligand to all 6 active sites with the difference spectrum for binding 1.4 ligand molecules to enzyme in which 4.6 of the active sites were already occupied. Difference spectra for the assembly of enzyme from subunits in the presence and absence of the bisubstrate analog were also measured. Since the difference spectrum for binding the last 2 ligand molecules to tetraliganded aspartate transcarbamoylase was almost identical with that for ligand binding to free catalytic subunit, subtraction of the assembly difference spectrum for unliganded T-state enzyme from the reconstitution difference spectrum for liganded R-state enzyme yielded directly the difference spectrum associated with the gross conformational change in the enzyme. Despite the binding of the bisubstrate analog in all 3 approaches, the final difference spectrum corresponds to the gross conformational change in unliganded aspartate transcarbamoylase.

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