Allosteric enzymes substrate binding

Biological processes involving protein-protein recognition are of special interest since they are the basis of many relevant events in the cellular machinery. Enzyme-substrate binding, protein membrane signaling, immunologic response, and protein quaternary structural changes caused by cofactors or allosteric modulators are only a few of the examples that show the importance of protein-protein interactions at the molecular level. 

The basic kinetic properties of this allosteric enzyme are clearly explained by combining Monod s theory and these structural results. The tetrameric enzyme exists in equilibrium between a catalytically active R state and an inactive T state. There is a difference in the tertiary structure of the subunits in these two states, which is closely linked to a difference in the quaternary structure of the molecule. The substrate F6P binds preferentially to the R state, thereby shifting the equilibrium to that state. Since the mechanism is concerted, binding of one F6P to the first subunit provides an additional three subunits in the R state, hence the cooperativity of F6P binding and catalysis. ATP binds to both states, so there is no shift in the equilibrium and hence there is no cooperativity of ATP binding. The inhibitor PEP preferentially binds to the effector binding site of molecules in the T state and as a result the equilibrium is shifted to the inactive state. By contrast the activator ADP preferentially binds to the effector site of molecules in the R state and as a result shifts the equilibrium to the R state with its four available, catalytically competent, active sites per molecule. 

Inhibition The decrease of the rate of an enzyme-catalyzed reaction by a chemical compound including substrate analogues. Such inhibition may be competitive with the substrate (binding at die active site of die enzyme) or non-competitive (binding at an allosteric site). 

Inhibition of a regulatory enzyme by a feedback inhibitor does not conform to any normal inhibition pattern, and the feedback inhibitor F bears little structural similarity to A, the substrate for the regulatory enzyme. F apparently acts at a binding site distinct from the substrate-binding site. The term allosteric is apt, because F is sterically dissimilar and, moreover, acts at a site other than the site for S. Its effect is called allosteric Inhibition. 

When binding of a substrate molecule at an enzyme active site promotes substrate binding at other sites, this is called positive homotropic behavior (one of the allosteric interactions). When this co-operative phenomenon is caused by a compound other than the substrate, the behavior is designated as a positive heterotropic response. Equation (6) explains some of the profile of rate constant vs. detergent concentration. Thus, Piszkiewicz claims that micelle-catalyzed reactions can be conceived as models of allosteric enzymes. A major factor which causes the different kinetic behavior [i.e. (4) vs. (5)] will be the hydrophobic nature of substrate. If a substrate molecule does not perturb the micellar structure extensively, the classical formulation of (4) is derived. On the other hand, the allosteric kinetics of (5) will be found if a hydrophobic substrate molecule can induce micellization. 

Activators and inhibitors regulate not the amount of enzyme protein but the activity ( efficiency ) of that which is present. Two principal mechanisms of control are (i) competitive and (ii) allosteric. Competitive control (inhibition) occurs when a compound which is structurally similar to the true substrate binds to the active site of the enzyme. This is how a number of drugs and poisons bring about their effect. For example, a group of therapeutic drugs called statins are used to treat heart disease because by inhibiting a key enzyme called HMGCoA reductase, they reduce the hepatic synthesis of cholesterol and therefore the plasma concentration of that lipid. 

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. 

PFK-1 is a classic example of a tetrameric allosteric enzyme. Each of the four subunits has two ATP binding sites one is the active site where ATP is co-substrate and the other is an inhibitory allosteric site. ATP may bind to the substrate (active) site when the enzyme is in either the R (active) or T (inhibited) form. The other co-substrate, F-6-P binds only to the enzyme in the R state. AMP may also bind to the R form and in so doing stabilises the protein in that active conformation permitting ATP and F-6-P to bind. 

Binding of a reversible inhibitor to an enzyme is rapidly reversible and thus bound and unbound enzymes are in equilibrium. Binding of the inhibitor can be to the active site, or to a cofactor, or to some other site on the protein leading to allosteric inhibition of enzyme activity. The degree of inhibition caused by a reversible inhibitor is not time-dependent the final level of inhibition is reached almost instantaneously, on addition of inhibitor to an enzyme or enzyme-substrate mixture. 

In addition to their active sites, these enzymes often have multiple sites for a variety of activators and inhibitors (e.g., AMP, ATP, citrate, fructose-2,6-bisphosphate [F2.6-BP]). Cooperative enzymes are sometimes referred to as allosteric enzymes because of the shape changes that are induced or stabilized by binding substrates, inhibitors, and activators. 

A subset in allosteric models of cooperativity. If an allosteric effector, upon binding to a cooperative enzyme, alters the Michaelis or dissociation constants (or [S0.5] value) for the substrate(s) (but not the h"max values), then that protein is a A system enzyme. See Monod-Wyman-Changeux Model K. E. Meet (1980) Meth. Enzymol. 64, 139. 

Non-competitive inhibitors. These inhibitors bind to the enzyme or the enzyme-substrate complex at a site other than the active site. This results in a decrease in the maximum rate of reaction, but the substrate can still bind to the enzyme. An analogous concept is that of allosteric inhibition. The site of binding of an allosteric inhibitor is distinct from the substrate binding site. In this case, the inhibitor is not a steric analog of the substrate and instead binds to the allosteric site (the phenomenon was termed thus by Monod and Jacob). 

The ability of proteins to exist in different conformations is termed allostery (see 2.2). Allosteric enzymes can assume various conformations which differ in catalytic activity and/or substrate binding capacity. 

High resolution X-ray structures of the a- and b- form of rabbit muscle phosphorylase permit a view into some of the structural differences of the various allosteric forms of the enzyme. Furthermore, the data give an impression of the mechanism of binding of effectors and the influence that phosphorylation has on substrate binding and enzyme activity (Barford et al., 1991). The following discussion will be restricted to the observed consequences of phosphorylation. 

---