2 Структура и функция белка (1160071), страница 38
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There are two major classes of regulatory enzymes in metabolic pathways. Allosteric enzymes function through reversible, noncovalent binding of a regulatory metabolite called a modulator. Theterm allosteric derives from Greek allos, "other," and stereos, "solid" or"shape." Allosteric enzymes are those having "other shapes" or conformations induced by the binding of modulators. The second class includes enzymes regulated by reversible covalent modification. Bothclasses of regulatory enzymes tend to have multiple subunits, and insome cases the regulatory site(s) and the active site are on separatesubunits.There are at least two other mechanisms by which enzyme activityis regulated.
Some enzymes are stimulated or inhibited by separatecontrol proteins that bind to them and affect their activity. Others areactivated by proteolytic cleavage, which unlike the other mechanismsis irreversible. Important examples of both these mechanisms arefound in physiological processes such as digestion, blood clotting, hormone action, and vision.COO"+ IH3N-C-HIL-ThreonineH-C-OHICH3,,threoninedehvdratasArBD+COO"IH3N—C-H* _ _ H—C—CH3 L-IsoleucineCH 2CH 3Figure 8-24 Feedback inhibition of the conversionof L-threonine into L-isoleucine, catalyzed by a sequence of five enzymes (Ei to E5). Threonine dehydratase (Ex) is specifically inhibited allosterically byL-isoleucine, the end product of the sequence, butnot by any of the four intermediates (A to D). Suchinhibition is indicated by the dashed feedback lineand the (g symbol at the threonine dehydratasereaction arrow.Part II Structure and Catalysis230No single rule governs the occurrence of different types of regulation in different systems.
To a degree, allosteric (noncovalent) regulation may permit fine-tuning of metabolic pathways that are requiredcontinuously but at different levels of activity as cellular conditionschange. Regulation by covalent modification tends to be all-or-none.However, both types of regulation are observed in a number of regulatory enzymes.Allosteric Enzymes Are Regulated byNoncovalent Binding of ModulatorsIn some multienzyme systems the regulatory enzyme is specificallyinhibited by the end product of the pathway, whenever the end productincreases in excess of the cell's needs.
When the regulatory enzymereaction is slowed, all subsequent enzymes operate at reduced ratesbecause their substrates are depleted by mass action. The rate of production of the pathway's end product is thereby brought into balancewith the cell's needs. This type of regulation is called feedback inhibition. Buildup of the pathway's end product ultimately slows the entire pathway.One of the first discovered examples of such allosteric feedbackinhibition was the bacterial enzyme system that catalyzes the conversion of L-threonine into L-isoleucine (Fig. 8-24).
In this system, thefirst enzyme, threonine dehydratase, is inhibited by isoleucine, theproduct of the last reaction of the series. Isoleucine is quite specific asan inhibitor. No other intermediate in this sequence of reactions inhibits threonine dehydratase, nor is any other enzyme in the sequenceinhibited by isoleucine. Isoleucine binds not to the active site, but toanother specific site on the enzyme molecule, the regulatory site. Thisbinding is noncovalent and thus readily reversible; if the isoleucineconcentration decreases, the rate of threonine dehydratase activityincreases. Thus threonine dehydratase activity responds rapidly andreversibly to fluctuations in the concentration of isoleucine in the cell.I Inactiveenzyme-M +MActiveenzyme-substratecomplexFigure 8—25 Schematic model of the subunit interactions in an allosteric enzyme, and interactionswith inhibitors and activators.
In many allostericenzymes the substrate binding site and the modulator binding site(s) are on different subunits, thecatalytic (C) and regulatory (R) subunits, respectively. Binding of the positive modulator (M) to itsspecific site on the regulatory subunit is communicated to the catalytic subunit through a conformational change. This change renders the catalyticsubunit active and capable of binding the substrate(S) with higher affinity. On dislocation of the modulator from the regulatory subunit, the enzyme reverts to its inactive or less active form.Allosteric Enzymes Are Exceptions to Many General RulesThe modulators for allosteric enzymes may be either inhibitory orstimulatory.
An activator is often the substrate itself, and regulatoryenzymes for which substrate and modulator are identical are calledhomotropic. When the modulator is a molecule other than the substrate the enzyme is heterotropic. Some enzymes have two or moremodulators.As already noted, the properties of allosteric enzymes are significantly different from those of simple nonregulatory enzymes discussedearlier in this chapter. Some of the differences are structural. In addition to active or catalytic sites, allosteric enzymes generally have oneor more regulatory or allosteric sites for binding the modulator (Fig.8-25). Just as an enzyme's active site is specific for its substrate, theallosteric site is specific for its modulator.
Enzymes with several modulators generally have different specific binding sites for each. Inhomotropic enzymes the active site and regulatory site are the same.Allosteric enzymes are also generally larger and more complexthan simple enzymes. Most of them have two or more polypeptidechains or subunits. Aspartate transcarbamoylase, which catalyzes thefirst reaction in the biosynthesis of pyrimidine nucleotides (Chapter21), has 12 polypeptide chains organized into catalytic and regulatoryChapter 8 Enzymessubunits. Figure 8-26 shows the quaternary structure of this enzyme,deduced from x-ray analysis.Other differences between nonregulated enzymes and allostericenzymes involve kinetic properties.
Allosteric enzymes show relationships between Vo and [S] that differ from normal Michaelis-Mentenbehavior. They do exhibit saturation with the substrate when [S] issufficiently high, but for some allosteric enzymes, when Vo is plottedagainst [S] (Fig. 8-27) a sigmoid saturation curve results, rather thanthe hyperbolic curve shown by nonregulatory enzymes. Although wecan find a value of [S] on the sigmoid saturation curve at which Vo ishalf-maximal, we cannot refer to it with the designation Km becausethe enzyme does not follow the hyperbolic Michaelis-Menten relationship. Instead the symbol [S]0.5 or K0m5 is often used to represent thesubstrate concentration giving half-maximal velocity of the reactioncatalyzed by an allosteric enzyme (Fig. 8-27).Sigmoid kinetic behavior generally reflects cooperative interactions between multiple protein subunits.
In other words, changes inthe structure of one subunit are translated into structural changes inadjacent subunits, an effect that is mediated by noncovalent interactions at the subunit-subunit interface. The principles are similar tothose discussed for cooperativity in oxygen binding to the nonenzymeprotein hemoglobin (p. 188). Homotropic allosteric enzymes generallyhave multiple subunits. In many cases the same binding site on eachsubunit functions as both the active site and the regulatory site. Thesubstrate can function as a positive modulator (an activator) becausethe subunits act cooperatively.
The binding of one molecule of the substrate to one binding site alters the enzyme's conformation and greatlyenhances the binding of subsequent substrate molecules. This accounts for the sigmoid rather than hyperbolic increase in Vo withincreasing [S].231Figure 8-26 The three-dimensional subunitarchitecture of the regulatory enzyme aspartatetranscarbamoylase; two different views. This allosteric regulatory enzyme has two catalytic clusters,each with three catalytic polypeptide chains, andthree regulatory clusters, each with two regulatorypolypeptide chains.
The catalytic polypeptides ineach cluster are shown in shades of blue and purple. Binding sites for allosteric modulators arefound on the regulatory subunits (shown in whiteand red). Modulator binding produces large changesin enzyme conformation and activity. The role ofthis enzyme in nucleotide synthesis, and details ofits regulation, will be discussed in Chapter 21.Figure 8-27 Substrate-activity curves for representative allosteric enzymes. Three examples ofcomplex responses given by allosteric enzymes totheir modulators, (a) The sigmoid curve given bya homotropic enzyme, in which the substrate alsoserves as a positive (stimulatory) modulator.
Notethat a relatively small increase in [S] in the steeppart of the curve can cause a very large increase inVo- Note also the resemblance to the oxygen-saturation curve of hemoglobin (see Fig. 7-28). (b) Theeffects of a positive modulator © , a negative modulator Q, and no modulator (5) on an allosteric enzyme in which K05 is modulated without a changein Vmax. (c) A less common type of modulation, inwhich Vmax is modulated with K05 nearly constant.232Part II Structure and CatalysisWith heterotropic enzymes, in which the modulator is a metaboliteother than the substrate itself, it is difficult to generalize about theshape of the substrate-saturation curve.
An activator may cause thesubstrate-saturation curve to become more nearly hyperbolic, with adecrease in K05 but no change in Vmax, thus resulting in an increasedreaction velocity at a fixed substrate concentration (Vo is higher forany value of [S]) (Fig. 8-27b). Other allosteric enzymes respond to anactivator by an increase in Vmax> with little change in K05 (Fig. 8-27c).A negative modulator (an inhibitor) may produce a more sigmoid substrate-saturation curve, with an increase in K05 (Fig.
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