Abstract
Although carbamoyl-phosphate synthetases (CPSs) share sequence identity, multidomain structure, and reaction mechanism, they have varying physiological roles and allosteric effectors. Escherichia coli CPS (eCPS) provides CP for both arginine and pyrimidine nucleotide biosynthesis and is allosterically regulated by metabolites from both pathways, with inhibition by UMP and activation by IMP and ornithine. The arginine-specific CPS from Saccharomyces cerevisiae (sCPS), however, apparently responds to no allosteric effectors. We have designed and analyzed a chimeric CPS (chCPS, in which the C-terminal 136 residues of eCPS were replaced by the corresponding residues of sCPS) to define the structural basis for the allosteric nonresponsiveness of sCPS and thereby provide insight into the mechanism for allosteric selectivity and responsiveness in the other CPSs. Surprisingly, ornithine and UMP each had a significant effect on chCPS activity, and did so at concentrations that were similar to those effective for eCPS. We further found that sCPS bound both UMP and IMP and that chCPS bound IMP, although none of these interactions led to changes in enzymatic activity. These findings strongly suggest that the nonresponsive sCPS is not able to communicate occupancy of the allosteric site to the active site but does contain a latent allosteric interaction domain.
Highlights
Escherichia coli and other enteric bacteria have a single enzyme that provides Carbamoyl phosphate (CP) for both arginine and pyrimidine biosynthesis
We further found that S. cerevisiae carbamoyl-phosphate synthetase (CPS) (sCPS) bound both UMP and IMP and that chimeric CPS (chCPS) bound IMP, none of these interactions led to changes in enzymatic activity
These findings strongly suggest that the nonresponsive sCPS is not able to communicate occupancy of the allosteric site to the active site but does contain a latent allosteric interaction domain
Summary
Materials—Oligonucleotides were synthesized at the Tufts University Analytical Core Facility. The plasmid pEK81, which expresses both the catalytic and regulatory chains of E. coli ATCase, and the E. coli strain EK1104, from which the genes encoding these subunits have been deleted, were gifts from Evan R. To create pES (the expression plasmid for chCPS), a 523-bp fragment encoding domain D of sCPS was amplified by PCR (pRS315/CPA2 as template, primer P12 introduced a SpeI site at nucleotide 3654 and P2 introduced a HindIII site and removed the existing SpeI site at nucleotide 3154) and digested with HindIII and SpeI to give a 506-bp fragment. Digestion of the modified pUCABI with HindIII and SpeI removed the 1468-bp fragment coding for domain D of eCPS and ornithine transcarbamoylase ( present in the original expression vector) and allowed ligation of the 506-bp CPA2 fragment. Enzyme Assays and Protein Analysis—CP synthesis was determined in a two-step assay by coupling the CPS reaction to that of ornithine transcarbamoylase and quantitating the resulting citrulline [20]. The multiple sequence alignment was produced by the Protein Information Resource (pir.georgetown.edu) and the three-dimensional visualization of the eCPS crystal structure (Swiss Protein Data base accession number 1CE8; Ref. 28) by Swiss-Model (GlaxoSmithKline)
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