Regulatory enzymes aspartate transcarbamoylase

FIGURE 6-27 Two views of the regulatory enzyme aspartate transcarbamoylase. (Derived from PDB ID 2AT2.)This allosteric regulatory... 

The first step in the pathway, formation of carbamoyl aspartate from aspartate and carbamoyl phosphate, is the primary regulatory point in the pathway. The enzyme, aspartate transcarbamoylase (ATCase) (see here), is activated by ATP and inhibited by CTP, which is the end product of the pathway. Another point of regulation is CTP synthetase, which is feedback inhibited by CTP and activated by GTP. In bacteria, synthesis of ATCase subunits is inhibited by high levels of UTP. The inverted regulatory effects of purine and pyrimidines in the pathway are yet another way cells maintain a proper balance of nucleotides. 

Aspartate transcarbamoylase (ATCase) from Escherichia coli is the most studied and best known regulatory enzyme. Yates and Pardee (1956) were the first to propose that the activity of ATCase is controlled by end product inhibition. This feedback inhibition was later studied in more detail by Gerhart and Pardee (1961, 1962, 1963). The three-dimensional structure of ATCase was determined by Lip-scombe and his coworkers [Wiley etal. (1971), Wiley and Lipscomb (1968), Warren etal. (1973)]. 

Control of enzymic activity arising from the modulated access of substrates to a channel leading to the active site. Such a scheme was suggested for aspartate carbamo-yltransferase which has its complement of active sites located on the interior surface of the complex comprised of catalytic and regulatory subunits. Nonetheless, isotope exchange studies of this enzyme suggest that this form of enzyme regulation does not apply in the case of aspartate transcarbamoylase . ... 

Allosteric enzymes are generally larger and more complex than nonallosteric enzymes. Most have two or more subunits. Aspartate transcarbamoylase, which catalyzes an early reaction in the biosynthesis of pyrimidine nucleotides (see Fig. 22-36), has 12 polypeptide chains organized into catalytic and regulatory subunits. Figure 6-27 shows the quaternary structure of this enzyme, deduced from x-ray analysis. 

A plot of VQ against [S] for an allosteric enzyme gives a sigmoidal-shaped curve. Allosteric enzymes often have more than one active site which co-operatively bind substrate molecules, such that the binding of substrate at one active site induces a conformational change in the enzyme that alters the affinity of the other active sites for substrate. Allosteric enzymes are often multi-subunit proteins, with an active site on each subunit. In addition, allosteric enzymes may be controlled by effector molecules (activators or inhibitors) that bind to a site other than the active site and alter the rate of enzyme activity. Aspartate transcarbamoylase is an allosteric enzyme that catalyzes the committed step in pyrimidine biosynthesis. This enzyme consists of six catalytic subunits each with an active site and six regulatory subunits to which the allosteric effectors cytosine triphosphate (CTP) and ATP bind. Aspartate transcarbamoylase is feedback-inhibited by the end-product of the pathway, CTP, which acts as an allosteric inhibitor. In contrast, ATP an intermediate earlier in the pathway, acts as an allosteric activator. 

Schematic representation of the subunit structure of aspartate transcarbamoylase and its dissociation into catalytic and regulatory subunits by mercurials, which can be further converted to inactive monomeric subunits by strong denaturing agents (e.g., sodium dodecyl sulfate). The native enzyme consists of two catalytic trimers placed one above the other, along with three dimeric regulatory subunits surrounding the catalytic trimers in an equatorial plane (a). Substrate maintains the enzyme in the catalytically more active relaxed (R) conformation, while cytidine tripho.sphate maintains it in the catalytically less active taut (T) conformation (b). [Reproduced, with permission from E. L. Smith, R. L. Hill,...

The answer is c. (Murray, pp 375-401. Scriver, pp 2513-2570. Sack, pp 121-138. Wilson, pp 287-320.) The steps of pyrimicfine nucleotide biosynthesis are summarized in the figure below. The first step in pyrimidine synthesis is the formation of carbamoyl phosphate. The enzyme catalyzing this step, carbamoyl phosphate synthetase (1), is feedback-inhibited by UMP through allosteric effects on enzyme structure (not by competitive inhibition with its substrates). The enzyme of the second step, aspartate transcarbamoylase, is composed of catalytic and regulatory subunits. The regulatory subunit binds CTP or ATP TTP has no role in the feedback inhibition of pyrimidine synthesis. Decreased rather than increased activity of enzymes 1 and 2 would be produced by allosteric feedback inhibition. 

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