Primary structure and properties of an inactive mutant aspartate transcarbamoylase.

A mutant form of Escherichia coli aspartate transcarbamoylase (ATCase) which lacks catalytic activity has been purified and characterized (Wall, K.A., Flatgaard, J.E., Gibbons, I., and Schachman, H.K. (1979) J. Biol. Chem 254, 11910-11916). Peptide mapping of the mutant and wild type catalytic chains followed by the determination of the amino acid sequence of the one altered peptide in the mutant indicated that a glycyl residue was replaced by aspartic acid. This substitution is located at position 125 in the tentative sequence kindly provided by W. Konigsberg (personal communication). The mutant protein has an overall secondary structure similar to that of the wild type as indicated by circular dichroism spectroscopy. However, marked changes in the reactivity of several amino acid residues were demonstrated. Lysyl residue 84 which in the wild type subunits reacts specifically with pyridoxal 5'-phosphate is only slightly reactive in the mutant even though the peptide containing that residue was not altered in amino acid composition. Another residue, cysteinyl 46, which is thought to be in the active site, is much more reactive toward p-hydroxymercuribenzoate in the mutant subunit than in the wild type protein. Finally, tyrosyl residue 213, which according to recent crystallographic studies is not near the active site and which exhibits an unusually low pK (9.1) in the wild type catalytic subunits, appears to have its pK shifted to 10.5 or higher as a result of the mutation. The evidence indicates that the substitution of an aspartyl for a glycyl residue at a region of the amino acid sequence remote from those residues in the active site causes sufficient modification of the tertiary structure to cause the loss of enzyme activity and to affect the reactivity of other residues in the protein. Moreover, the quaternary structure of the intact enzyme is altered as well since the subunit interactions are greatly weakened.