Enzymes allosteric effects

Enzyme inhibition. The enzymes of biotransformation may be inhibited by a single exposure to chemicals. This occurs by several mechanisms formation of a complex, competition between substrates, destruction of the enzyme, reduced synthesis of the enzyme, allosteric effects, and lack of cofactors. The consequences will depend on the role of metabolism in toxicity in the same way as induction (see above). 

Ghanges in the availability of substrates are responsible for most changes in metabolism either directly or indirectly acting via changes in hormone secretion. Three mechanisms are responsible for regulating the activity of enzymes in carbohydrate metabolism (1) changes in the rate of enzyme synthesis, (2) covalent modification by reversible phosphorylation, and (3) allosteric effects. 

Competitive, non-competitive and allosteric effects all rely on weak binding between the enzyme and the various ligands (small molecules which attach to a bigger molecule). Such weak binding attractions are not covalent bonds, unlike the situation described below. 

Some enzymes are controlled by both allosterism and covalent modification often brought about by hormone stimulation of the cell. Allosteric effects will take effect immediately because the enzyme is responding to local intracellular conditions of substrate or coenzyme concentrations, but covalent effects because they are driven by hormonal stimulation may take a little longer to have an impact but will be part of a coordinated response in several tissues of the body sensitive to the hormone. 

The key control enzyme in the pathway is glycogen synthase (GS) which occurs in either a high activity state (GS-a) or a low activity state (GS-b) the switch from one to the other is brought about partly by covalent modification of the enzyme in response to stimulation by glucagon and partly by allosteric effects of key metabolites. Glycogen... 

There are many examples of phosphorylation/dephosphorylation control of enzymes found in carbohydrate, fat and amino acid metabolism and most are ultimately under the control of a hormone induced second messenger usually, cytosolic cyclic AMP (cAMP). PDH is one of the relatively few mitochondrial enzymes to show covalent modification control, but PDH kinase and PDH phosphatase are controlled primarily by allosteric effects of NADH, acetyl-CoA and calcium ions rather than cAMP (see Table 6.6). 

The ability of a reversible inhibitor to alter substrate affinity on binding to the enzyme, and vice versa, as well as the ability of the inhibitor to alter fcp, generally occur because of allosteric effects. As the allosteric effects of a molecule are related to its size, shape, and charge distribution, it might be expected that different... 

Allosteric inhibitors bind to a separate binding site outside the active center (6). This results in a conformational change in the enzyme protein that indirectly reduces its activity (see p. 116). Allosteric effects practically only occur in oligomeric enzymes. The kinetics of this type of system can no longer be described using the simple Micha-elis-Menten model. 

Depending on the enzyme, allosteric effectors can influence the maximum rate V ax. the semi-saturation concentration [A]o.5, and the Hill coef dent h. If it is mainly V ax that is changed, the term V system is used. Much more common are K systems , in which allosteric effects only influence [A]o.5 and h. 

ID-myo-inositol 1,3,4,5-tetrakisphosphate <1> (<1> recombinant, catalytically active fragment of isoform C, allosteric product activation in the absence of Ca /calmodulin, which per se activates enzyme and abolishes the allosteric effect [31]) [31]... 

An alkaline pyrophosphatase from rat liver cytoplasm has been partially purified and characterized (24) the corresponding enzyme from mice is inhibited by Mg J+-ADP and free PPj, and free Mg2+ has been implicated as an allosteric activator (23). Partial heat inactivation results in loss of the apparent allosteric effects. Rat liver mitochondrial pyrophosphatase, which is inhibited by adenine nucleotides (36), appears to be bound to the inside of the inner mitochondrial membrane (37). This enzyme, after solubilization, has been separated into two fractions which have somewhat different specificity (24, 38). A pyrophosphatase strongly simulated by sulfhydryl reagents (39) has been partially purified from brain tissue (40). The mono-magnesium PPj complex appears to be the true substrate for this enzyme (41). Pynes and Younathan have purified a pyrophosphatase 1800-fold from human erythrocytes (43). The properties of this enzyme are strikingly similar to those of the yeast enzyme the major difference appears to be the more rigid substrate specificity of the erythrocyte enzyme in the presence of Znz. ... 

In the presence of sodium dodecyl sulfate (SDS), the apparent molecular weight was approximately 25,000, consistent with the presence of four subunits. The dissociation of the enzyme into half-molecules was also observed at alkaline pH (83). At pH 9.0 this dissociation was promoted by the addition of AMP in the cold and prevented by the addition of FDP. It was proposed that dissociation at alkaline pH was an extreme manifestation of a conformational change induced by AMP which was related to the allosteric effects observed with this effector. In this connection it is noteworthy that the enzyme desensitized to AMP inhibition by treatment with I2 or FDNB was also no longer susceptible to dissociation by AMP. 

The manner in which the reduction of ribonucleotides to deoxyribonucleotides is regulated has been studied with reductases from relatively few species. The enzymes from E. coli and from Novikoff s rat liver tumor have a complex pattern of inhibition and activation (fig. 23.25). ATP activates the reduction of both CDP and UDP. As dTTP is formed by metabolism of both dCDP and dUDP, it activates GDP reduction, and as dGTP accumulates, it activates ADP reduction. Finally, accumulation of dATP causes inhibition of the reduction of all substrates. This regulation is reinforced by dGTP inhibition of the reduction of GDP, UDP, and CDP and by dTTP inhibition of the reduction of the pyrimidine substrates. Because evidence suggests that ribonucleotide reductase may be the rate-limiting step in deoxyribonucleotide synthesis in at least some animal cells, these allosteric effects may be important in controlling deoxyribonucleotide synthesis. 

Allosteric effects play a major role in biology, for instance in the conformational changes induced by the binding of an effector and regulating the activity of an enzyme [9.14a] they have also been studied in synthetic receptors [8.201, 8.211, 9.14b, c]. Similarly, cooperativity, a thermodynamically well defined process [9.15a], is displayed by a number of biological species as well as by abiotic ones [8.70a, 8.201, 9.15b-d] (see also Section 9.3.1, [9.64, 9.65]), in particular in organized media such as polymer solutions or gels [7.8cd, 8.290, 8.292]. 

In addition to being easier to fit than the hyperbolic Michaelis-Menten equation, Lineweaver-Burk graphs clearly show differences between types of enzyme inhibitors. This will be discussed in Section 4.5. However, Lineweaver-Burk equations have their own distinct issues. Nonlinear data, possibly indicating cooperative multiunit enzymes or allosteric effects, often seem nearly linear when graphed according to a Lineweaver-Burk equation. Said another way, the Lineweaver-Burk equation forces nonlinear data into a linear relationship. Variations of the Lineweaver-Burk equation that are not double reciprocal relationships include the Eadie-Hofstee equation7 (V vs. V7[S]) (Equation 4.14) and the Hanes-Woolf equation8 ([S]/V vs. [S]) (Equation 4.15). Both are... 

Partially purified maize endosperm ADPGlc PPase (34U/mg) was found to be activated by 3PGA and Fru 6-P (25- and 17-fold, respectively) and inhibited by Pi.77 The heterotetrameric endosperm enzyme has been cloned and expressed in E. coli, and its regulatory properties were compared to an isolated allosteric mutant less sensitive to Pi inhibition.117 As indicated above, the increase of starch noted in the mutant maize endosperm ADPGlc PPase insensitive to Pi inhibition supports the importance of the allosteric effects of 3-PGA and Pi in vivo. Also as indicated above, it is believed that the major endosperm ADP-Glc PPase isoform is located in the cytosol.141... 

The cooperative binding of O2 by hemoglobin and the allosteric effects in many enzymes require interaction between sites that are widely separated in space. The MWC model was proposed in 1965 to incorporate allosteric and conformational effects in an explanation of enzyme cooperativity. The seminal observation was that most cooperative proteins have several identical subunits (protomers) in each molecule (oligomer) this situation is imperative for binding cooperativity. The MWC model is defined as follows ... 

A considerable expansion of the use of the spin-state equilibrium as a probe of allosteric effects is desirable. Thus it should be possible to use it in assisting studies on oxidases such as trytophan pyrolase and peroxidase which are mainly high-spin iron(III) enzymes. In a later section we shall show how the discussion can be extended to proteins containing haem a. 

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