Genetic characterization of the folding domains of the catalytic chains in aspartate transcarbamoylase.

In Salmonella typhimurium strains which produce high constitutive levels of aspartate transcarbamoylase due to the pyrH700 mutation, the bulk of the carbamoyl phosphate of the cell is consumed for the biosynthesis of pyrimidines. As a consequence, there is little substrate available for arginine synthesis and the cell growth is impeded. Suppression of arginine auxotrophy by mutations which block aspartate transcarbamoylase activity provides a positive selection technique for mutant strains defective in this enzyme activity. A genetic analysis was performed on 29 mutant strains harboring defects in the structural gene pyrB, encoding the catalytic chains of aspartate transcarbamoylase of Escherichia coli. Extracts from 15 strains contained intact, inactive enzyme-like molecules of the same size as the purified wild type enzyme. These same extracts contained a predominant polypeptide chain which migrated electrophoretically at the same rate as catalytic chains from wild type enzyme. In addition to these 15 different missense mutants, 14 others (presumably chain-terminating mutants) were isolated; no polypeptides corresponding to full length catalytic chains were detected in these strains. Based on their reversion and suppression properties, seven were designated as frameshift and two as amber nonsense. A fine structure recombination map of the pyrB locus was constructed from a series of three-factor transductional crosses. Mutational sites were correlated with regions in the polypeptide sequence by relating their map positions to that of mutation pyrB231 which results in an amino acid replacement at position 128. Moreover, since recent crystallographic studies indicate that residue 128 is located near the junction between the NH2- and COOH-terminal folding domains, the mutational sites can be placed within either of these two regions of tertiary structure. Interallelic complementation experiments showed four units of complementation. Those defining the alpha and beta units were missense mutants with their mutational sites in the NH2- and COOH-terminal domains, respectively. The mutants determining the delta and gamma units involved premature polypeptide chain termination and their mutational sites were correlated with distal regions of the two respective domains. Several mutants of the chain-terminating type failed to complement members of more than one unit. Possible effects of the various mutations and their implications for mechanisms of complementation and enzyme activity are presented.