The Behavior of Allosteric Enzymes

Signals regulate the flow of traffic in much the same fashion as control mechanisms in chemical reactions. 

What is the concerted model for allosteric behavior What is the sequential model for allosteric behavior  

Does phosphorylation always increase enzyme activity  

How does the architecture of the active site affect catalysis  

How do the critical amino acids catalyze the chymotrypsin reaction  

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Two theoretical models that attempt to explain the behavior of allosteric enzymes are the concerted model and the sequential model. In the concerted (or symmetry) model, it is assumed that the enzyme exists in only two states T(aut) and R(elaxed). Substrates and activators bind more easily to the R conformation, whereas inhibitors favor the T conformation. The term concerted is applied to this model because the conformations of all the protein s protomers are believed to change simultaneously when the first effector binds. (This rapid concerted change in conformation maintains the protein s overall symmmetry.) The binding of an activator shifts the equilibrium in favor of the R form. An inhibitor shifts the equilibrium toward the T conformation. 

The key to allosteric behavior, including cooperativity and modifications of cooperativity, is the existence of multiple forms for the quaternary structures of allosteric proteins. The word allosteric is, derived from alio, other, and stetic, shape, referring to the fact that the possible conformations affect the behavior of the protein. The binding of substrates, inhibitors, and activators changes the quaternary structure of allosteric proteins, and the changes in structure are reflected in the behavior of those proteins. A substance that modifies the quaternary structure, and thus the behavior, of an allosteric protein by binding to it is called an allosteric effector. The term effector can apply to substrates, inhibitors, or activators. Several models for the behavior of allosteric enzymes have been proposed, and it is worthwhile to compare them. 

The two principal models for the behavior of allosteric enzymes are the concerted model and the sequential model. They were proposed in 1965 and 1966, respectively, and both are currently used as a basis for interpreting experimental results. The concerted model has the advantage of comparative simplicity, and it describes the behavior of some enzyme systems very well. 

Recall Does the behavior of allosteric enzymes become more or less cooperative in the presence of inhibitors ... 

Recall Distinguish between the concerted and sequential models for the behavior of allosteric enzymes. 

Reflect and Apply Is it possible to envision models for the behavior of allosteric enzymes other than the ones that we have seen in this chapter ... 

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. 

When ATGase catalyzes the condensation of aspartate and carbamoyl phosphate to form carbamoyl aspartate, the graphical representation of the rate as a function of increasing substrate concentration (aspartate) is a sigmoidal curve rather than the hyperbola obtained with nonallosteric enzymes (Figure 7.2a). The sigmoidal curve indicates the cooperative behavior of allosteric enzymes. In this two-substrate reaction, aspartate is the substrate for which the concentration is varied, while the concentration of carbamoyl phosphate is kept constant at high levels. 

Recall How is the cooperative behavior of allosteric enzymes reflected in a plot of reaction rate against substrate concentration ... 

Not all enzymes follow Michaelis-Menten kinetics. The kinetic behavior of allosteric enzymes does not obey the Michaelis-Menten equation. 

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. 

The Kinetic Properties of Allosteric Enzymes Diverge from Michaelis-Menten Behavior... 

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. 

We ve been working on the behavior of zinc in enzymes and also what are called allosteric enzymes, that is, enzymes that undergo conformational change when they have different subunits, different proteins, to make a... 

Allosteric interactions control the behavior of proteins through reversible changes in quaternary structure, but this mechanism, effective though it may be, is not the only one available. A zymogen, an inactive precursor of an enzyme, can be irreversibly transformed into an active enzyme by cleavage of covalent bonds. 

How does an organism ensure that glycogen synthesis and glycogen breakdown do not operate simultaneously If this were to occur, the main result would be the hydrolysis of UTP, which would waste chemical energy stored in the phosphoric anhydride bonds. A major controlling factor lies in the behavior of glycogen phosphorylase. This enzyme is subject not only to allosteric control but also to another control feature covalent modification. We saw an earlier example of this kind of control in the sodium-potassium pump in Section 8.6. In that example, phosphorylation and dephosphorylation of an enzyme determined whether it was active, and a similar effect takes place here. 

MgCl (10 mM) markedly increased the affinity of malic enzyme for NADP (Kj = 0.38 mM), without significant change in the cooper at ivity (n = 1.57). L-malate not only highly decreased the value to 0.11 mM but also notoriously affected the allosteric behavior, as revealed by an njj value of 0.18 (Table 1). Contrarily to this findings, cooperati-vity was absent in saturation isotherms for NADP obtained from kinetic studies (Figure 2B). From these hyperbolic patterns (n = 1) a K value for NADP of 0.020 mM was calculated (Table 1). ... 

We have observed a stimulation of ADP-ribosylation by nanomolar concentrations of benzamides both in intact and permeabilized cells. This stimulation is reminiscent of that found with competitive inhibitors of allosteric enzymes. At very low NAD " concentrations, Kun has reported finding a sigmoidal curve of velocity versus NAD concentration for purified nuclear ADP-ribosyl transferase (12). The observed allosteric behavior is likely to arise from the interaction of NAD binding sites either on the same or different enzyme molecules. There is no evidence that nuclear ADP-ribosyl transferase is an oligomeric protein. More than one nuclear ADP-ribosyl transferase molecule is involved however in the... 

A very distintive characteristic of the regulatory phenomenon already described is that the observed changes of the kinetic parameters require the integrity of the membrane, which means that the modification of the fatty acid composition change the behavior of the enzyme when membrane lipids are structuraly organized. This work shows that complementary information could be obtained from the studies of allosteric behavior and temperature-dependent activity... 

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