Regulatory enzymes feedback control

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)]. 

Many regulatory enzymes are allosterically controlled by a product or products of the reaction sequence that is to say they are subject to feedback control. The binding of an effector to the enzyme causes a conformational change which is transmitted to the active site and causes either an increase or a decrease in activity over a range of substrate concentrations. Frequently, in the absence of an activator or in the presence of an inhibitor, the enzyme exhibits sigmoidal kinetics with respect to substrate concentration. Addition of the activator or removal of the inhibitor restores normal saturation kinetics. 

The two-step reduction of HMG-CoA to mevalonate (Fig. 22-1, step a)n 15 is highly controlled, a major factor in regulating cholesterol synthesis in the human liver.121617 The N-terminal portion of the 97-kDa 888-residue mammalian FlMG-CoA reductase is thought to be embedded in membranes of the ER, while the C-terminal portion is exposed in the cytoplasm.16 Tire enzyme is sensitive to feedback inhibition by cholesterol (see Section D, 2). The regulatory mechanisms include a phosphorylation-dephosphorylation cycle and control of both the rates of synthesis and of proteolytic degradation of this key en-... 

Allosteric control involves the reversible binding of a compound to an allosteric site referred to as a regulatory site on the enzyme. These compounds may be either one of the compounds involved in the metabolic pathway (feedback regulators) or a compound that is not a product of the metabolic pathway. In both cases, the binding usually results in conformational changes, which either activate or deactivate the enzyme. Proenzymes also act as a form of enzyme control. 

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 this experiment we will examine some of the properties of the aspartate transcarbamylase of Escherichia coli, which is typical of many enzymes subject to feedback inhibition and which has been studied extensively. Aspartate transcarbamylase (ATCase) catalyzes the first reaction unique to the biosynthesis of pyrimidine nucleotides. ATCase is subject to specific inhibition by quite low concentrations of one of its end products, cytidine 5 -triphosphate (CTP). This relationship and two other regulatory interactions important to the control of pyrimidine biosynthesis are summarized in Figure 9-1. 

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