Allosteric enzymes Aspartate transcarbamoylase

Fig. 3. Plot of initial reaction velocity (V0) against substrate concentration for the allosteric enzyme aspartate transcarbamoylase.

In another important allosteric enzyme, aspartate transcarbamoylase, studies at 3.0 and 2.8 A have indicated, inter alia, the binding site for CTP. ... 

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

Whereas substrates bind to the active sites of enzymes, other nonsubstrate molecules (allosteric modulators) bind to the allosteric sites. The significance and role of the allosteric site is well illustrated by the example of the enzyme aspartate transcarbamoylase, which catalyzes reaction 1. 

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. 

Although the Michaelis-Menten model provides a very good model of the experimental data for many enzymes, a few enzymes do not conform to Michaelis-Menten kinetics. These enzymes, such as aspartate transcarbamoylase (ATCase), are called allosteric enzymes (see Topic C5). 

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. 

Aspartate transcarbamoylase (aspartate carbamoyltransferase ATCase), a key enzyme in pyrimidine biosynthesis (see Topic FI), provides a good example of allosteric regulation. ATCase catalyzes the formation of N-carbamoylaspar-tate from aspartate and carbamoyl phosphate, and is the committed step in pyrimidine biosynthesis (Fig. 2). The binding of the two substrates aspartate and carbamoyl phosphate is cooperative, as shown by the sigmoidal curve of V0 against substrate concentration (Fig. 3). 

Aspartate transcarbamoylase (ATCase) is an allosteric enzyme of the bacterium Escherichia coli, which has been extensively studied. This enzyme catalyzes the transfer of the carbamoyl group from carbamoyl phosphate to the amino group of aspartate ... 

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. 

This discussion of functional domains within an allosteric protein can be extended to the case of heteropolymeric allosteric enzymes. Consider aspartate transcarbamoylase... 

This is the first step of the pathway that leads to the formation of cytosine, a building block for DNA synthesis. The form of cytosine that is used to synthesize DNA (and RNA) is the molecule cytidine triphosphate (CTP). When intracellular CTP concentrations are high, CTP molecules bind more often to the allosteric sites on aspartate transcarbamoylase molecules, causing a change in the shape of the enzyme that slows reaction 1 down markedly. Thus, CTP is an allosteric inhibitor of this enzyme. 

The catalytic activity of enzymes is controlled in several ways. Reversible allosteric control is especially important. For example, the first reaction in many biosynthetic pathways is allosterically inhibited by the ultimate product of the pathway. The inhibition of aspartate transcarbamoylase by cyti-dine triphosphate (Section 10.1) is a well-understood example of feedback inhibition. This type of control can be almost instantaneous. Another recurring mechanism is reversible covalent modification. For example, glycogen phosphorylase, the enzyme catalyzing the breakdown of glycogen, a storage form of sugar, is activated by phosphorylation of a particular serine residue when glucose is scarce (Section 21.2.1). 

The difference between the velocity curves for chymotrypsin and aspartate transcarbamoylase demonstrates the difference between an allosteric enzyme and a nonallosteric enzyme. 

The reaction of carbamoyl phosphate with aspartate to produce W-carbamo-ylaspartate is the committed step in pyrimidine biosynthesis. The compounds involved in reactions up to this point in the pathway can play other roles in metabolism after this point, A -carbamoylaspartate can be used only to produce pyrimidines—thus the term committed step. This reaction is catalyzed by aspartate transcarbamoylase, which we discussed in detail in Ghapter 7 as a prime example of an allosteric enzyme subject to feedback regulation. The next step, the conversion of A-carbamoylaspartate to dihydroorotate, takes place in a reaction that involves an intramolecular dehydration (loss of water) as well as cyclization. This reaction is catalyzed by dihydroorotase. Dihydroorotate is converted to orotate by dihydroorotate dehydrogenase, with the concomitant conversion of NAD to NADH. A pyrimidine nucleotide is now formed by the reaction of orotate with PRPP to give orotidine-5 -monophosphate (OMP), which is a reaction similar to the one that takes place in purine salvage (Section 23.8). Orotate phosphoribosyltransferase catalyzes this reaction. Finally, orotidine-5 -phosphate decarboxylase catalyzes the conversion of OMP to UMP... 

Aspartate transcarbamoylase (ATCase, EC 2.1.3.2) is an allosteric enzyme which controls the first step of p5oimidine de novo biosynthesis. It catalyzes the condensation of L-aspartic acid with carbamyl phosphate to produce carbamoyl-L-aspartate... 

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