Enzymes, active conformation allosteric

PFK-1 is a classic example of a tetrameric allosteric enzyme. Each of the four subunits has two ATP binding sites one is the active site where ATP is co-substrate and the other is an inhibitory allosteric site. ATP may bind to the substrate (active) site when the enzyme is in either the R (active) or T (inhibited) form. The other co-substrate, F-6-P binds only to the enzyme in the R state. AMP may also bind to the R form and in so doing stabilises the protein in that active conformation permitting ATP and F-6-P to bind. 

Figure 3.14 Diagram illustrating regulation of enzyme activity by an allosteric regulator. Note the representation of the conformational change.

But where there is an equilibrium among two or more conformations of the enzyme in solution, crystallization may select out only one of the conformations. a-Chymotrypsin has a substantial fraction of an inactive conformation present under the conditions of crystallization, but only the active form of the enzyme crystallizes. An allosteric effector molecule that changes the conformation of the protein in solution may have no effect on the crystalline protein, as, for example, with phosphorylase b.5A The enzyme is frozen in one conformation, with the crystal lattice forces preventing any conformational change. On the other hand, the addition of an effector to phosphorylase a causes the crystals first to crack and then to anneal, giving crystals of the enzyme in a second conformation. 

The concept of control of metabolic activity by allosteric enzymes or the control of enzyme activity by ligand-induced conformational changes arose from the study of metabolic pathways and their regulatory enzymes. A good example is the multi-enzymatic sequence catalysing the conversion of L-threonine to L-isoleucine shown in Fig. 5.32. 

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. 

In addition, allosteric enzymes may be controlled by effector molecules (activators and inhibitors) that bind to the enzyme at a site other than the active site (either on the same subunit or on a different subunit), thereby causing a change in the conformation of the active site which alters the rate of enzyme activity (cf. the binding of C02, H+ and 2,3-bisphosphoglycerate to hemoglobin see Topic B4). An allosteric activator increases the rate of enzyme activity, while an allosteric inhibitor decreases the activity of the enzyme. 

The enzyme phosphofructokinase is allosteric, that is, it is made up of equivalent units that possess specific reaction sites for the fixation of the substrate and product. Each unit exists in two conformational states one active with more affinity for the substrate, and one inactive. The reaction products of phosphofructokinase (FDP and ADP) displace the conformational equilibrium in favor of the active form of the enzyme. This may create a destabilizing effect on the excess entropy production. In the glycolytic cycle, the allosteric properties of the phosphofructokinase may lead to oscillations. Consider the following simple model... 

The addition of small molecules has been shown to change the enantioselectivity of certain enzyme-catalyzed reactions. It is believed that such molecules bind to a site in the protein different from the active site, which leads to a conformational change in the active site. Such enzymes are called allosteric enzymes, i.e. enzymes that comprise of multiple subunits and multiple active sites. Binding of a cosubstrate or small molecule may cause an increase or decrease in the activity or selectivity of the enzyme. 

There are two major ways of control. One mechanism involves reversible covalent modifications, such as phosphorylation dephosphorylation, the other requires conformational transitions by binding an allosteric ligand or regulator protein. It follows an example of regulation of an enzyme, of which the activity is subject to control by both mechanisms, then we compare the regulation of an enzyme with regulation of components of cellular signalling pathways, of which many have no enzymic activity. 

N is often limiting in the marine environment. Further, many enzymes are sensitive to cellular substrate concentrations rather than extracellular concentrations and it is difficult to measure the relevant intracellular metabohte pools. In vitro assays may affect the conformation of enzymes and the degree to which they are modified. For example, allosteric effects (see Section 1.3.3) may be modified under in vitro conditions. Many enzymes undergo posttranslational regulation wherein enzyme activity is affected by binding of activator/inactivator proteins and covalent modification of the enzyme (e.g., adenylylation, phosphorylation or carbamylation) (Ottaway, 1988). When there is posttranslational modification of enzymes, enzyme activity measured in assays may be unrelated to in vivo activity (see Section 2.2.1) and there are few ways to determine the extent of enzyme modification in nature. 

Non-competitive inhibitors reversibly to somewhere other than the active site they change the protein conformation allosterically, and reduce the rate at which the enzyme turns over product. They have no effect on Km as the active-site of uninhibited enzyme molecules will only encounter substrate, and no unproductive binding will occur. They do however reduce the apparent Vm consequently the apparent Vm will be reduced, since the protein is no longer as enzymatically competent. Such inhibitors are generally not substrate analogues. 

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