Allosteric enzymes regulatory

Two other allosteric enzyme regulatory reactions also help to regulate glycolysis the conversion of fructose 6-phosphate to fructose 1,6-diphosphate by phos-phofructokinase and the conversion of phosphoenolpyru-vate to pyruvate by pyruvate kinase. 

If the kinetics of the reaction disobey the Michaelis-Menten equation, the violation is revealed by a departure from linearity in these straight-line graphs. We shall see in the next chapter that such deviations from linearity are characteristic of the kinetics of regulatory enzymes known as allosteric enzymes. Such regulatory enzymes are very important in the overall control of metabolic pathways. 

Regulatory or allosteric enzymes like enzyme 1 are, in some instances, regulated by activation. That is, whereas some effector molecules such as F exert negative effects on enzyme activity, other effectors show stimulatory, or positive, influences on activity. 

The working hypothesis is that, by some means, interaction of an allosteric enzyme with effectors alters the distribution of conformational possibilities or subunit interactions available to the enzyme. That is, the regulatory effects exerted on the enzyme s activity are achieved by conformational changes occurring in the protein when effector metabolites bind. 

The metabolic control is exercised on certain key regulatory enzymes of a pathway called allosteric enzymes. These are enzymes whose catalytic activity is modulated through non-covalent binding of a specific metabolite at a site on the protein other than the catalytic site. Such enzymes may be allosterically inhibited by ATP or allosterically activated by ATP (some by ADP and/or AMP). 

In die metabolic pathway to an amino add several steps are involved. Each step is die result of an enzymatic activity. The key enzymatic activity (usually die first enzyme in the synthesis) is regulated by one of its products (usually die end product, eg die amino add). If die concentration of die amino add is too high die enzymatic activity is decreased by interaction of die inhibitor with the regulatory site of die enzyme (allosteric enzyme). This phenomenon is called feedback inhibition. 

In addition to the binding of substrate (or in some cases co-substrates) at the active site, many enzymes have the capacity to bind regulatory molecules at sites which are usually spatially far removed from the catalytic site. In fact, allosteric enzymes are invariably multimeric (i.e. have a quaternary structure) and the allosteric (regulatory) sites are on different subunits of the protein to the active site. In all cases, the binding of the regulatory molecules is non covalent and is described in kinetic terms as noncompetitive inhibition. 

One excellent example of a Emax-type allosteric enzyme is Escherichia coli phosphoglycerate dehydrogenase (PGDH), a tetramer of identical subunits that catalyzes the formation of D-3-phosphohydroxypyruvate from D-3-phosphoglycerate in a reaction that uses NAD+ as a redox cofactor. This regulatory enzyme is allosteri-cally controlled by serine. All available information suggests that the effects on the for substrate are minor... 

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. 

Allosteric enzymes show relationships between V0 and [S] that differ from Michaelis-Menten kinetics. They do exhibit saturation with the substrate when [S] is sufficiently high, but for some allosteric enzymes, plots of V0 versus [S] (Fig. 6-29) produce a sigmoid saturation curve, rather than the hyperbolic curve typical of non-regulatory enzymes. On the sigmoid saturation curve we can find a value of [S] at which V0 is half-maximal, but we cannot refer to it with the designation Km, because the enzyme does not follow the hyperbolic Michaelis-Menten relationship. Instead, the symbol [S]0 e or K0,5 is often used to represent the substrate concentration giving half-maximal velocity of the reaction catalyzed by an allosteric enzyme (Fig. 6-29). 

Homotropic allosteric enzymes generally are multisubunit proteins and, as noted earlier, the same binding site on each subunit functions as both the active site and the regulatory site. Most commonly, the substrate acts as a positive modulator (an activator), because the subunits act cooperatively the binding of one molecule... 

The activity of allosteric enzymes is adjusted by reversible binding of a specific modulator to a regulatory site. Modulators may be the substrate itself or some other metabolite, and the effect of the modulator may be inhibitory or stimulatory. The kinetic behavior of allosteric enzymes reflects cooperative interactions among enzyme subunits. 

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. 

Altering the control of metabolic pathways can also be achieved by genetic manipulation. As proteins are generated using the template stored as a fragment of DNA, the structure of the allosteric enzyme may be altered so that there is little or no regulatory control. It is therefore possible to generate mutants that over-produce metabolites, and techniques based on this principle have been most widely exploited in amino-acid and nucleotide production 2 . 

All biosynthetic pathways are under regulatory control by key allosteric enzymes that are influenced by the end products of the pathways. For example, the first step in the pathway for purine biosynthesis is inhibited in a concerted fashion by nucleotides of either adenine or guanine. In addition, the nucleoside monophosphate of each of these bases inhibits its own formation from inosine monophosphate (IMP). On the other hand, adenine nucleotides stimulate the conversion of IMP into GMP, and GTP is needed for AMP formation. 

There are exceptions to Michaelis-Menten behaviour. For example allosteric enzymes which instead of a hyperbolic curve in a Lversus [S] graph yield a sigmoidal plot (the behaviour is rather like non-catalytic allosteric proteins, such as haemoglobin, Section 2.5. This type of curve can indicate cooperative binding of the substrate to the enzyme. We have discussed cooperativity in Section 1.5 (see also Section 10.4.3). In addition, regulatory molecules can further alter the activity of allosteric enzymes. 

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. 

The enzyme responsible for this step is fructose 1,6-bisphosphatase. Like its glycolytic counterpart, it is an allosteric enzyme that participates in the regulation of gluconeogenesis. We will return to its regulatory properties later in the chapter. 

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