Allostery and cooperativity in multimeric proteins: bond-to-bond propensities in ATCase

Aspartate carbamoyltransferase (ATCase) is a large dodecameric enzyme with six active sites that exhibits allostery: its catalytic rate is modulated by the binding of various substrates at distal points from the active sites. A recently developed method, bond-to-bond propensity analysis, has proven capable of predicting allosteric sites in a wide range of proteins using an energy-weighted atomistic graph obtained from the protein structure and given knowledge only of the location of the active site. Bond-to-bond propensity establishes if energy fluctuations at given bonds have significant effects on any other bond in the protein, by considering their propagation through the protein graph. In this work, we use bond-to-bond propensity analysis to study different aspects of ATCase activity using three different protein structures and sources of fluctuations. First, we predict key residues and bonds involved in the transition between inactive (T) and active (R) states of ATCase by analysing allosteric substrate binding as a source of energy perturbations in the protein graph. Our computational results also indicate that the effect of multiple allosteric binding is non linear: a switching effect is observed after a particular number and arrangement of substrates is bound suggesting a form of long range communication between the distantly arranged allosteric sites. Second, cooperativity is explored by considering a bisubstrate analogue as the source of energy fluctuations at the active site, also leading to the identification of highly significant residues to the T↔R transition that enhance cooperativity across active sites. Finally, the inactive (T) structure is shown to exhibit a strong, non linear communication between the allosteric sites and the interface between catalytic subunits, rather than the active site. Bond-to-bond propensity thus offers an alternative route to explain allosteric and cooperative effects in terms of detailed atomistic changes to individual bonds within the protein, rather than through phenomenological, global thermodynamic arguments.

Effects of salts on the aspartate transcarbamylase of a halophilic eubacterium, Vibrio costicola

The aspartate transcarbamylase (ATCase) in cell-free extracts of the moderately halophilic eubacterium, Vibrio costicola, was stable in 1.5 M NaCl, but not in 0.5 M NaCl on prolonged storage at 4 °C in concentrated extracts. At lower salt concentrations, activity was lost rapidly. ATCase activity was optimal at about 1.5 M NaCl or 1.0 M KCl, although high activity was detected at 0.15 M NaCl. In the presence of 0.03 M aspartate both succinate and maleate inhibited ATCase activity. CTP inhibited the activity of the enzyme at low salt concentrations (0.15 to 0.3 M). Much less inhibition occurred at higher salt concentrations. Precipitating the enzyme with ammonium sulphate resulted in loss of CTP inhibition. The ATCase of V. costicola differs from those of a nonhalophile (Saccharomyces cerevisiae) and an extremely halophilic archaebacterium (Halobacterium cutirubrum) in its salt-response patterns of activity and regulation.Key words: halophilic, aspartate transcarbamylase, Vibrio costicola.

Escherichia coli aspartate transcarbamylase: a novel marker for studies of gene amplification and expression in mammalian cells.

Eucaryotic expression vectors containing the Escherichia coli pyrB gene (pyrB encodes the catalytic subunit of aspartate transcarbamylase [ATCase]) and the Tn5 phosphotransferase gene (G418 resistance module) were transfected into a mutant Chinese hamster ovary cell line possessing a CAD multifunctional protein lacking ATCase activity. G418-resistant transformants were isolated and analyzed for ATCase activity, the ability to complement the CAD ATCase defect, and the ability to resist high concentrations of the ATCase inhibitor N-(phosphonacetyl)-L-aspartate (PALA) by amplifying the donated pyrB gene sequences. We report that bacterial ATCase is expressed in these lines, that it complements the CAD ATCase defect in trans, and that its amplification engenders PALA resistance. In addition, we derived rapid and sensitive assay conditions which enable the determination of bacterial ATCase enzyme activity in the presence of mammalian ATCase.

Escherichia coli aspartate transcarbamylase: a novel marker for studies of gene amplification and expression in mammalian cells

Eucaryotic expression vectors containing the Escherichia coli pyrB gene (pyrB encodes the catalytic subunit of aspartate transcarbamylase [ATCase]) and the Tn5 phosphotransferase gene (G418 resistance module) were transfected into a mutant Chinese hamster ovary cell line possessing a CAD multifunctional protein lacking ATCase activity. G418-resistant transformants were isolated and analyzed for ATCase activity, the ability to complement the CAD ATCase defect, and the ability to resist high concentrations of the ATCase inhibitor N-(phosphonacetyl)-L-aspartate (PALA) by amplifying the donated pyrB gene sequences. We report that bacterial ATCase is expressed in these lines, that it complements the CAD ATCase defect in trans, and that its amplification engenders PALA resistance. In addition, we derived rapid and sensitive assay conditions which enable the determination of bacterial ATCase enzyme activity in the presence of mammalian ATCase.

Nuclear localization of aspartate transcabamoylase in Saccharomyces cerevisiae.

The cytochemical technique using the in situ precipitation of orthophosphate ions liberated specifically by the aspartate carbamoyltransferase (ATCase) (EC 2.1.3.2) reaction indicated that in Saccharomyces cerevisiae this enzyme is confined to the nucleus. This observation is in accordance with the result reported by Bernhardt and Davis (1972), Proc. Natl. Acad. Sci. U. S. A. 69:1868-1872) on Neurospora crassa. The nuclear compartmentation was also observed in a mutant strain lacking proteinase B activity. This finding indicates that this proteinase is not involved in the nuclear accumulation of ATCase, and that the activity observed in the nucleus corresponds to the multifunctional form associated with the uracil path-specific carbamoylphosphate synthetase and sensitive to feedback inhibition by UTP. In a ura2 strain transformed by nonintegrated pFL1 plasmids bearing the URA2-ATCase activity encoding gene, the lead phosphate precipitate was observed predominantly in the cytoplasm. This finding enhances the reliability of the technique used by eliminating the possibility of an artifactual displacement of an originally cytoplasmic reaction product during the preparation of the material for electron microscopy. On the other hand, nuclei isolated under hypoosmotic conditions do not exhibit the ATCase activity that is recovered in the cytosolic fractions after differential centrifugation of the lysate in Percoll gradient. A release of the protein from the nuclei during the lysis step, consistent with its nucleoplasmic localization, is postulated.

Heat-induced disaggregation of a multifunctional enzyme complex catalyzing the first steps in pyrimidine biosynthesis in bakers' yeast

A highly purified enzyme complex (from bakers' yeast), which possesses carbamylphosphate synthetase (CPSase) and aspartate transcarbamylase (ATCase) activities and a regulatory site for both activities at which feedback inhibition is exerted by UTP, was subjected to heat at 50 °C, and all three of the above parameters were followed. A treatment of 5 min was sufficient to cause disaggregation of the complex (original molecular weight 600 000) and appearance of subunits of approximately one-quarter the size of the unheated material. These subunits possessed only the ATCase activity, which was insensitive to uridine triphosphate; no component possessing CPSase activity was found in the sucrose density gradient. The ATCase was relatively resistant to heat, but there was a rapid and roughly parallel decline in the CPSase and in the sensitivity of the ATCase to inhibition by UTP. However, the residual CPSase activity, at any duration of heating, remained fully sensitive to inhibition by uridine triphosphate. CPSase activity was optimal at temperatures between 25° and 30° whereas the ATCase followed the Arrhenius law to 37 °C. Two alternative hypotheses of the structure of the enzyme complex were put forward to account for these data. Both of these hypotheses require that CPSase activity be a function of the oligomeric aggregate and not of individual subunits.