Allosteric enzymes interaction

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. 

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. 

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. 

C. Many allosteric enzymes have multiple subunits whose interaction accounts for their unusual kinetic properties. 

The oligomycin inhibition requires interaction of F, with other polypeptide chains of the coupling factor that are associated with the membrane used for reconstitution. In a sense this is quite analogous to allosteric enzymes, where regulation is achieved by modulation of inter-subunit interactions. 

As with other multisubunit enzymes (e.g., allosteric enzymes), the structural integrity of a membrane-bound enzyme primarily is maintained by noncovalent interactions such as hydrogen bonding, electrostatics, and hydrophobic interactions. Hydrophobic polypeptides (or hydrophobic portions of polypeptides) apparently are used to anchor the enzymes to the membrane through interactions with phospholipids. Therefore, I would characterize the interaction between the enzyme and membrane as chemical in nature rather than as geometric. ... 

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. 

One indication of the importance of intersubunit interactions in allosteric enzymes is that many such enzymes do not obey the classical Michaelis-Menten kinetic equation. A... 

Is the interaction between an allosteric effector and an allosteric enzyme always an equilibrium ... 

Studies of the salt and temperature dependence of fibrinopep-tide release show that electrostatic interactions allow thrombin to bind at diffusion-controlled rates (De Gristofaro and Di Cera, 1992 Vindigni and Di Gera, 1996). After thrombin is bound, hydrophobic interactions result in a conformational change in this allosteric enzyme, converting it to a faster form with a higher catalytic efficiency (Guinto et al, 1995). 

Figure 22 Examples of enzyme kinetic plots used for determination of Km and Vmax for a normal and an allosteric enzyme Direct plot [(substrate) vs. initial rate of product formation] and various transformations of the direct plot (i.e., Eadie-Hofstee, Lineweaver-Burk, and/or Hill plots) are depicted for an enzyme exhibiting traditional Michaelis-Menten kinetics (coumarin 7-hydroxylation by CYP2A6) and one exhibiting allosteric substrate activation (testosterone 6(3-hydroxylation by CYP3A4/5). The latter exhibits an S-shaped direct plot and a hook -shaped Eadie-Hofstee plot such plots are frequently observed with CYP3A4 substrates. Km and Vmax are Michaelis-Menten kinetic constants for enzymes. K is a constant that incorporates the interaction with the two (or more) binding sites but that is not equal to the substrate concentration that results in half-maximal velocity, and the symbol n (the Hill coefficient) theoretically refers to the number of binding sites. See the sec. III.C.3 for additional details.

The term "quaternary structure" refers to the interaction of several polypeptide chains in a noncovalent manner to form multisubunit protein particles termed oligomers. Individual subunit polypeptide chains are also referred to as protomers. Oligomers usually have an even number of subunits (two or more). The noncovalent interactions may be of the hydrophobic, hydrogen bond, or the polar type. Examples are hemoglobin and lactate dehydrogenase (four protomers each) and many allosteric enzymes. 

Allosteric Effectors. Many enzymes are subject to metabolic regulation through interaction with metabolites that often act at allosteric sites, which are distinct from the active site. The kinetic behavior of such enzymes is often more complex than the behavior we have discussed above, and such complex kinetics may serve as an indication that you are dealing with an allosteric enzyme. Further discussion of this subject is found in Experiments 9 and 15. 

Two major models for allosteric enzymes have been proposed. These are the sequential interaction model and the concerted-symmetry ... 

Figure 4-50 The sequential interaction model of allosteric enzymes. As each site is occupied, the subunit carrying the site undergoes a change from the A conformation to the B conformation. As a result, new interactions between subunits are established and the affinities of the vacant sites change. K represents a dissociation constant. Thus, if the affinities of vacant sites increase, a, b, and c (the interaction factors) are <1 and we observe positive cooperativity (a sigmoidal velocity curve). The sequential interaction model also provides for, negative cooperativity (a, b, and c are > ). (o ) Dimer model. The two ways of arranging S to form a singly-occupied species is shown, (fe) Tetramer model. For simplicity, only one arrangement of each occupied species is shown.

Schematic diagram of conformational changes of. sequentially induced-fit model for a dimeric allosteric enzyme. The TT conformation is progressively converted to the RR conformation via the intermediate TR conformation through cooperative interaction in the presence of the positive modulator. In the presence of the negative modulator, the opposite conformational changes occur. In this model, the notion of. symmetry is discarded and the concept of induced fit is emphasized.

Allosteric enzymes are oligomeric proteins exhibiting a sigmoidal dependence of the reaction rate on substrate concentration instead of a normal , hyperbolic (classical Michaelis-Menten) one. At first the rate increases only slightly with increasing substrate concentration there is then a rapid increase until near the maximum rate. This behaviour results from the presence of a regulatory allosteric center located on the same or another subunit as the catalytic center. This center may interact... 

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