Long range effects of amino acid substitutions in the catalytic chain of aspartate transcarbamoylase. Localized replacements in the carboxyl-terminal α-helix cause marked alterations in allosteric properties and intersubunit interactions

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A single α-helical polypeptide segment of 21 amino acids near the carboxyl terminus of the catalytic chain of aspartate transcarbamoylase from Escherichia coli has been shown recently to be important for the in vivo folding of the chains and assembly of the enzyme (Peterson, C. B., and Schachman, H. K. (1991) Proc. Natl. Acad. Sci. U. S. A. 88, 458-462). Calorimetric measurements on purified mutant enzymes showed that single amino acid replacements within this secondary structural element affect the overall thermal stability of the oligomeric enzyme and the energetics of the interactions between polypeptide chains within the holoenzyme. Studies presented here demonstrate that marked changes in cooperativity occur due to single amino acid substitutions. Replacement of Gln288 by either Ala or Glu leads to a striking increase in the Hill coefficient of the holoenzymes and a substantial increase in the aspartate concentration corresponding to one-half V(max). In contrast, the isolated catalytic trimers harboring these same substitutions were similar in activity to the wild-type subunit, with the same affinity for aspartate as indicated by the values of K(m). Substituting Ala for the only charged residue in the helix, Arg296, caused a marked reduction in enzyme activity, as well as a greatly reduced stability of the holoenzyme due to a substantial weakening of the interactions between the catalytic and regulatory subunits. A subunit exchange method was used to demonstrate the changes in interchain interactions resulting from the amino acid substitutions and to show the additional weakening upon the binding of the bisubstrate ligand, N-(phosphonacetyl)-L-aspartate, at the active sites. Taken together, the results on this series of mutant enzymes illustrate how the effects of single amino acid replacements in one element of secondary structure are propagated throughout the molecule to positions remote from the site of the substitution.

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Journal of Biological Chemistry

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