Effects of Replacement of Active Site Residue Glutamine 231 on Activity and Allosteric Properties of Aspartate Transcarbamoylase

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Since crystallographic studies on Escherichia coli aspartate transcarbamoylase (ATCase) indicate that Gin 231 is in the active site of the enzyme and participates in the binding of the substrate, aspartate, it seemed of interest to examine mutant enzymes in which Gin 231 was replaced by Asn or lie. The two mutant forms containing amino acid substitutions were characterized by a combination of steady-state kinetics, hydrodynamic measurements, and equilibrium ligand binding techniques. Both mutant forms exhibited a dramatic reduction in the affinity of the protein for substrates and substrate analogues as well as a very large decrease in catalytic activity. Moreover, the amino acid substitutions introduced within the active site of the enzyme led to unusual allosteric properties in the mutant enzymes. Although the bisubstrate analogue N-(phosphonoacetyl)-L-aspartate promotes the characteristic global conformational change in the mutant forms that is observed with the wild-type enzyme, the combination of substrate and substrate analogue does not. Cooperativity with respect to substrate binding is largely reduced compared to wild-type ATCase. Also, the effector molecules ATP and CTP which bind to the regulatory chains have dramatic effects on the activity of the mutant enzymes containing replacements for Gin 231 in the catalytic chains. In stark contrast to the wild-type enzyme, in which effects of nucleotides are manifested primarily by changes in the K0.5 of the enzyme, ATP and CTP have large effects on the Vmax of the mutant enzymes. Analysis of equilibrium binding of the analogue N-(phosphonoacetyl)-L-aspartate to the mutant enzymes based on the two-state model of Monod, Wyman, and Changeux provided insight into the unusual allosteric properties of the mutant forms of ATCase. © 1992, American Chemical Society. All rights reserved.

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