Co-operative interactions between the catalytic sites in Escherichia coli aspartate transcarbamylase. Role of the C-terminal region of the regulatory chains |
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Authors: | X G Xi F Van Vliet M M Ladjimi B De Wannemaeker C De Staercke A Piérard N Glansdorff G Hervé R Cunin |
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Institution: | Laboratoire d'Enzymologie, C.N.R.S., Gif-sur-Yvette, France. |
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Abstract: | In aspartate transcarbamylase (ATCase) each regulatory chain interacts with two catalytic chains each one belonging to a different trimeric catalytic subunit (R1-C1 and R1-C4 types of interactions as defined in Fig. 1). In order to investigate the interchain contacts that are involved in the co-operative interactions between the catalytic sites, a series of modified forms of the enzyme was prepared by site-directed mutagenesis. The amino acid replacements were devised on the basis of the previously described properties of an altered form of ATCase (pAR5-ATCase) which lacks the homotropic co-operative interactions between the catalytic sites. The results obtained (enzyme kinetics, bisubstrate analog influence and pH studies) show that the R1-C4 interaction is essential for the establishment of the enzyme conformation that has a low affinity for aspartate (T state), and consequently for the existence of co-operativity between the catalytic sites. This interaction involves the 236-250 region of the aspartate binding domain of the catalytic chain (240s loop) and the 143-149 region of the regulatory chain which comprises helix H3'. |
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