Enzymes allosteric systems

In contrast to the kinetics of isosteric (normal) enzymes, allosteric enzymes such as ACTase have sigmoidal (S-shaped) substrate saturation curves (see p. 92). In allosteric systems, the enzyme s af nity to the substrate is not constant, but depends on the substrate concentration [A]. Instead of the Michaelis constant Km (see p. 92), the substrate concentration at half-maximal rate ([AJo.s) is given. The sigmoidal character of the curve is described by the Hill coef cient h. In isosteric systems, h = 1, and h increases with increasing sigmoid icity. 

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. 

Cooperative enzyme systems in which the presence of an allosteric effector results in an alteration of the ymax value of the system (as opposed to changes in the value(s) K systems). See Vmax-Type (or, V-Type) Allosteric System Allosterism... 

S.G Rhee, R. Park, P.B. Chock, and E.R. Stadtman. 1978. Allosteric regulation of monocyclic interconvertible enzyme cascade systems Use of Escherichia coli glutamine synthetase as an experimental model Proc. Natl. Acad. Sci. USA 75 3138-3142. (PubMed)... 

Segel, 1. H., Emyme Kinetics Behavior and Analysis of Rapid Equilibrium and Steady-State Enzyme Systems. Wiley-Interscience (1975). This book starts at the same elementary level as Biochemical Calculations and progresses to the modern subjects of steady-state kinetics of mullireac-tant enzymes, allosteric enzymes, isotope exchange, and membrane transport. 

Fig. 2.10. Scheme of an autocatalytic enzyme reaction. The allosteric enzyme catalysing the reaction is formed by several subunits (not shown), which exist in the conformational states R and T. These states differ in their affinity for the substrate and/or in their catalytic activity. In the model by Monod et al. (1965), the transition between the two conformational states is concerted for all subunits. Here the product is a positive effector, i.e. an activator, as it binds in an exclusive or preferential manner to the most active, R state, of the enzyme. The system is open as the substrate S is injected at a constant rate while the product P disappears as a result of its utilization in a subsequent enzyme reaction. 

Enzyme stimulation is an increase in enzyme activity resrrlting directly from the addition of a chemical. This is a somewhat imusual phenomenon in ertzymology, usually relegated to classically allosteric systems [200]. The concept is that a chemical stimulates the catalytic activity of an enzyme. This cooperativity may be considered in two aspects. One is homotropic cooperativity, in which a chemical stimulates its own biotransformation. This is usually manifested in sigmoidal (S-shaped) plots of v versus S. Heterotropic cooperativity is the stimulation of catalytic activity by direct addition of a different compound. 

For many years hemoglobin was the only allosteric protein whose stereochemical mechanism was understood in detail. However, more recently detailed structural information has been obtained for both the R and the T states of several enzymes as well as one genetic repressor system, the trp-repressor, described in Chapter 8. We will here examine the structural differences between the R and the T states of a key enzyme in the glycolytic pathway, phosphofructokinase. 

First draw both Lineweaver-Burk plots and Hanes-Woolf plots for the following a Monod-Wyman-Changeux allosteric K enzyme system, showing separate curves for the kinetic response in (1) the absence of any effectors (2) the presence of allosteric activator A and (3) the presence of allosteric inhibitor I. Then draw a similar set of curves for a Monod-Wyman-Changeux allosteric Uenzyme system. 

Because this enzyme catalyzes the committed step in fatty acid biosynthesis, it is carefully regulated. Palmitoyl-CoA, the final product of fatty acid biosynthesis, shifts the equilibrium toward the inactive protomers, whereas citrate, an important allosteric activator of this enzyme, shifts the equilibrium toward the active polymeric form of the enzyme. Acetyl-CoA carboxylase shows the kinetic behavior of a Monod-Wyman-Changeux V-system allosteric enzyme (Chapter 15). 

When the initial LA concentration is large, the quantity of substrate transferred to the aqueous phase allows the lipoxygenation to progress. This reaction consumes LA and produces HP, which favor the transfer of residual substrate between the two phases. Then catalysis and transfer have a reciprocal influence on each other. We demonstrated that the use of a non-allosteric enzyme in a compartmentalized medium permits the simulation of a co-operativity phenomenon. The optimal reaction rate in the two-phase system is reached for a high initial LA concentration 14 mM. Inhibition by substrate excess is observed in two-phase medium. 

Manipulation of one enzymatic step in a system can have wide reaching consequences because of the interplay between metabolite levels and a wide range of regulatory circuits. These circuits can operate at the level of transcription, translation, post-translational modification, or through allosteric and competitive influences on the kinetic properties of enzymes. 

In some cases, an inhibitor can bind to more than one site on an enzyme protein, with inhibition resulting from binding at multiple sites. Binding affinities at the two (or more) sites may be different, and mechanisms of inhibition may be dilferent for example, high-affinity inhibition might occur through an allosteric site and lower affinity inhibition through the active site. Analysis of such systems is complex and may require a combination of several of the approaches outlined later. 

It is not a requirement that binding of an allosteric modulator to an enzyme must result in inhibition of activity indeed, in some mixed inhibition systems described earlier, both a and P have values between 0 and 1. If an increase in substrate affinity outweighs a decrease in at lower substrate concentrations, an increase in enzyme activity may occur, relative to control values, and the use of the term inhibitor to classify such a compound is open to debate. 

The indirect correlation is the major source of cooperativity in biochemical systems, such as hemoglobin (Chapter 6) or allosteric enzymes (Chapter 8). The model treated in this section is the simplest binding model having indirect correlation. We now examine some of the outstanding properties of the indirect correlation... 

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