Aspartate carbamoyltransferase catalytic subunit

Figure 7-20 (A) Subunit assembly of two C3 catalytic trimers (green) and three R2 regulatory dimers around the periphery in aspartate carbamoyltransferase. After Krause et a/.109 Courtesy of William N. Lipscomb. The aspartate-and carbamoylphosphate-binding domains of the catalytic subunits are labeled Asp and CP, respectivley, while the zinc and allosteric domains of the regulatory subunits are labeled Alio and Zn, respectively. (B) Ribbon drawing of a single pair of regulatory (left) and catalytic (right) subunits with the structural domains labeled. MolScript drawing from Thomas et al.no...

Aspartate carbamoyltransferase catalyzes the formation of carbamoyl aspartate from carbamoyl phosphate and aspartate in the first committed step of pyrimidine biosynthesis (Chap. 15). The enzyme from the bacterium E. coli (Mr = 310,000) consists of 12 subunits, six regulatory and six catalytic. CTP is a negative effector i.e., it inhibits the enzyme, and does so through binding to the regulatory subunits. ATP is a positive effector that acts through the regulatory subunits, while succinate inhibits the reaction by direct competition with aspartate at the active site (see Chap. 9 for more on effectors). 

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. 

Figure 23-11 The difference spectrum for the catalytic subunit of aspartate carbamoyltransferase (Fig. 7-20) in the presence and absence of succinate and carbamoyl phosphate. (A) The spectrum of the unperturbed enzyme (points) fitted with two log normal curves (solid line). (B) The difference between the spectrum of enzyme plus 0.09 M succinate and 4.3 mM carbamoyl phosphate and that shown in (A) (compare with published difference spectrum for intact aspartate carbamoyltransferase). ...

Bacterial aspartate carbamoyltransferase is subject to allosteric inhibition [92] by CTP, one of the end products of the pathway. The enzyme from E. coli has been extensively studied because of its regulatory properties [92-94]. The native molecule consists of three regulatory subunits and two catalytic ones. However, three different classes of aspartate carbamoyltransferase have been recognized in different bacterial species, differing in molecular size and kinetic properties [95]. The enzyme has also been demonstrated in a number of animal tissues [96] and together with carbamoyl phosphate synthetase II and dihydro-orotase was found in... 

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