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Five mutant versions of aspartate transcarbamylase have been isolated, all with single amino acid substitutions in the catalytic chain of the enzyme. A previously isolated pyrB nonsense mutant was suppressed with supB, supC, supD and supG to create enzymes with glutamine, tyrosine, serine or lysine, respectively, inserted at the position of the nonsense codon. Each of these enzymes was purified to homogeneity and kinetically characterized. The approximate location of the substitution was determined by using tryptic fingerprints of the wild-type enzyme and the enzyme obtained with a tyrosine residue inserted at the position of the nonsense codon. By first cloning the pyrBI operon, from the original pyrB nonsense strain, followed by sequencing of the appropriate portion of the gene, the exact location of the mutation was determined to be at position 209 of the catalytic chain. Site-directed mutagenesis was used to generate versions of aspartate transcarbamylase with tyrosine and glutamic acid at this position. The Tyr209 enzyme is identical with that obtained by suppression of the original nonsense mutation with supC. The two enzymes produced by site-directed mutagenesis were purified using a newly created overproducing strain. Kinetic analysis revealed that each mutant has an altered affinity for aspartate, as judged by variations in the substrate concentration at one-half maximal activity. In addition, the mutants exhibit altered Hill coefficients and maximal activities. In the wild-type enzyme, position 209 is a tryptophan residue that is involved in the stabilization of a bend in the molecule near the subunit interface region. The alteration in homotropic cooperativity seems to be due to changes induced in this bend in the molecule, which stabilizes alternate conformational states of the enzyme.

A pyrimidine auxotroph of Escherichia coli was isolated which contained a defect in its ability to synthesize both oroate phosphoribosyl transferase, the product of the gene pyrE, and orotidine monophosphate decarboxylase, product of the gene pyrF. A single location on the E. coli linkage map was found to be responsible for the loss of both enzyme activities. This gene was located near cysE at 80.55 min by a combination of Hfr crosses and P1 transductions. The pyrimidine requirement was also corrected by episome F'140 which was found not to carry any pyrimidine structural genes. These data confirm the existence of a new gene, pyrS, unlinked to any previously mapped pyrimidine structural gene, responsible for partial control of pyrimidine biosynthesis. A spontaneous revertant of the mutant strain was also identified which displayed constitutive levels of aspartate transcarbamylase, dihydroorotase, dihydroorotate dehydrogenase, orotidine monophosphate decarboxylase, and limited levels of orotate phosphoribosyl transferase. A model is proposed in which the pyrS gene product is an activator protein, necessary for the transcription of the pyrE and pyrF genes. This activator protein is nonfunctional in the original mutant strain, and partially functional in the revertant strain. The data presented here cannot rule out an alternative mechanism involving a repressor.

A strain of Escherichia coli has been constructed which greatly overproduces the enzyme aspartate transcarbamylase. This strain has a deletion in the pyrB region of the chromosome and also carries a leaky mutation in pyrF. Although this strain is a pyrimidine auxotroph, it will grow very slowly without pyrimidines if a plasmid containing the pyrB gene is introduced into it. Derepression occurs when this strain exhausts its uracil supply during exponential growth. Under extreme derepression, aspartate transcarbamylase can account for as much as 60% of the total cellular protein. This host strain/plasmid system can be utilized for the rapid purification of wild-type aspartate transcarbamylase or plasmid-born mutant versions of the enzyme. This system is particularly well-suited for analysis of the latter since the control of overproduction resides exclusively on the bacterial chromosome. Therefore, any plasmid bearing the pyrBI operon can be made to overproduce aspartate transcarbamylase in this host strain. Based on this system, a rapid purification procedure has been developed for E. coli aspartate transcarbamylase. The purification scheme involves an ammonium sulfate fractionation followed by a single precipitation of the enzyme at its isoelectric point. In a similar fashion, this strain can also be employed to produce exclusively the catalytic subunit of the enzyme if the plasmid only carries the pyrB gene. This system may be adapted to overproduce other proteins as well by using this host strain and the strong pyrB promoter linked to another gene.