Aspartate transcarbamylase catalytic subunit

Figure II-4 Examples of the quaternary structure of proteins, (a) A drawing of glutamine synthetase of coli showing the orientation of the 12 identical subunits of the enzyme. (b) A drawing of aspartate transcarbamylase of coli showing the proposed orientation of the 6 catalytic subunits (labeled C, each MW = 33,000), and 6 regulatory subunits (labeled R, each MW =...

Three-dimensional structure of E. coli aspartate transcarbamylase. Half of the native c6r6 molecule (see Fig. II-4) is shown. The catalytic (c) submit, which binds the substrates aspartate and carbamyl phosphate is shown in light shading. The regulatory (r) subunit, which binds the allosteric effectors CTP and ATP, is shown in dark shading. From Kantrowitz, E.R., et al. (1980). E. coli Aspartate Transcarbamylase. Part 11 Structure and Allosteric Interactions. Trends Biochem Sci 5 150 and Stryer, L. (1995). Biochemistry, 4th ed. New York Freeman, Figure 10-5, p. 240. Reprinted by permission. 

Before it is used by the class, the extract should be tested for overproduction of ATCase by assay see General Procedure for the Assay of Aspartate Transcarbamylase Activity, step 9 Range Finding below. The extract can also be examined by SDS-PAGE for overproduction of the ATCase catalytic and regulatory subunits (34 kDa and 19 kDa, respectively). To do this, use the procedure described in Experiment 4 for 15% gels. 

Nowlan, S. F., and Kantrowitz, E. R. (1985). Superproduction and Rapid Purification of Escherichia coli Aspartate Transcarbamylase and Its Catalytic Subunit under Extreme Derepression of the Pyrimidine Pathway. J Biol Chem 260 14712. 

Describe at least one gene you would expect to be able to clone using the following genes as bait in a yeast two-hybrid experiment alpha-globin the catalytic subunit of protein kinase A and the catalytic subunit of aspartate transcarbamylase. 

Site-directed mutagenesis has been used to establish that the active site lies at the interface between subunits of certain oligomeric enzymes (62-64). The analysis relies on restoration of activity on forming a hybrid from proteins containing mutations at two positions. Studies of this type were first performed on aspartate transcarbamylase (aspartate carbamoyltransferase) (62, 63), where an active hybrid catalytic trimer was isolated from a mixture of two inactive mutants. The rationale for this analysis is shown in Fig. 8, illustrating wodc done on ribulose-bisphosphate carboxylase (64). Two mutant enzymes, eaeh unable to carry out catalysis, were recombined to form hybrids. Based on random association of monomers to form the catalytic dimer as shown in Fig. 8, it is expected that 50% of the trimers should form one wild-type active site (B, C), such that the mixture of the hybrids exhibits 25% of the wild-type activity. This complementation demonstrates that the active site must be at the interface between the subunits. 

In this situation, two different polypeptide chains interact to form a new specific complex, whose biologic activity can be modified by ligands that bind to the precursor subunits. This type of regulation has been extensively studied for the aspartate transcarbamylase of E. coli (Gerhart, 1970). This enzyme catalyzed the initial step in the synthesis of cytidine nucleotides it is allosterically inhibited by CTP and shows positive cooperativity for substrate. It may be dissociated by mercurials into catalytic subunits, which are insensitive to CTP and which exhibit hyperbolic kinetics for substrate, and into regulatory subunits which bind CTP. 

The trimeric catalytic subunit assembly of aspartate transcarbamylase can be reversibly unfolded by GuHCl (Ghelis and Herve, 1978). Up to 95% of the activity is restored under suitable conditions (i.e., 25°C, pH 7.0). 

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