ALLOSTERIC ENZYMES: CHARACTERISTICS, MECHANISMS OF ACTION, EXAMPLES - BIOLOGY - 2025

An allosteric enzyme (from the Greek: allo, different + stereos, three-dimensional space) is a protein in which indirect interactions occur between topographically different sites, by the binding of substrates and regulatory molecules (ligands).

The binding of a ligand to a specific site is influenced by the binding of another effector ligand (or modulator ligand) to a different (allosteric) site on the enzyme. This is known as allosteric interactions, or cooperative interactions.

Example of an enzyme. Source: Thomas Shafee

When the effector ligand increases the binding affinity of another ligand to the enzyme, cooperativity is positive. When the affinity decreases, cooperativity is negative. If two identical ligands participate in the cooperative interaction, the effect is homotropic, and if the two ligands are different, the effect is heterotropic.

Cooperative interaction produces reversible changes in the molecular structure of the enzyme, at the tertiary and quaternary structure level. These changes are known as conformational changes.

History

The concept of allosteric interaction emerged more than 50 years ago. It has evolved through time, namely:

-In 1903, the sigmoidal curve of binding of hemoglobin to oxygen was observed.

-In 1910, the sigmoidal curve of O ₂ binding to hemoglobin was mathematically described using Hill's equation.

-In 1954, Novick and Szilard showed that an enzyme located at the beginning of a metabolic pathway was inhibited by the end product of this pathway, which is known as negative feedback.

-In 1956, Umbarger discovered that L-threonine deaminase, the first enzyme in the L-isoleucine biosynthesis pathway, was inhibited by L-isoleucine, and that it did not exhibit typical Michaelis-Menten kinetics with a hyperbolic curve, rather it had a sigmoidal curve.

-In 1963, Perutz et al., Discovered through X-rays conformational changes in the structure of hemoglobin when it binds to oxygen. Monod and Jacob renamed the regulatory sites "allosteric sites."

-In 1965, Monod, Wyman and Changeux proposed the symmetric model, or MWC model (initial letters of Monod, Wyman and Changeux) to explain allosteric interactions.

-In 1966, Koshland, Nemethy and Filmer proposed the sequential or induced coupling model, or KNF model, to explain allosteric interactions.

-In 1988, the X-ray structure of aspartate transcarbamylase demonstrated the symmetric model postulated by Monod, Wyman and Changeux.

-In the 1990s, mutations, covalent modifications and pH changes were considered as allosteric effectors.

-In 1996, the X-ray structure of the lac repressor demonstrated allosteric transitions.

Mechanisms of action and examples

-Characteristics of the MWC and KNF models of allosteric regulation

MWC model

The original hypothesis of the MWC model proposed the following (Monod, Wyman, Changeux, 1965)

Allosteric proteins are oligomers made up of symmetrically related protomers. Protomers are made up of polypeptide chains or subunits.

The oligomers have at least two conformation states (R and T). Both states (of the quaternary structure) spontaneously establish an equilibrium, with or without bound ligand.

When the transition from one state to another occurs, symmetry is preserved, and the affinity of a site (or several) stereospecific sites for a ligand is altered.

In this way, the cooperative binding of the ligands follows from the cooperative interaction between subunits.

KNF model

The KNF model hypothesis proposed the following (Koshland, Nemethy, Filmer, 1966): Ligand binding produces a change in tertiary structure in a subunit. This change in conformation affects neighboring subunits.

The binding affinity of the protein ligand depends on the number of ligands that it holds together. Thus, allosteric proteins have multiple conformational states that include intermediate states.

During the last five decades, the MWC and KNF models have been evaluated through biochemical and structural studies. It was shown that numerous allosteric proteins, including enzymes, comply with what is proposed in the MWC model, although there are exceptions.

The MWC model and allosteric enzymes (or allosteric regulatory enzymes)

Allosteric enzymes are often larger and more complex than non-allosteric enzymes. Aspartate transcarbamylase (Asp transcarbamylase or ATCase) and phosphofructokinase-1 (PFK-1) are classic examples of allosteric enzymes that comply with the MWC model.

AT House of

ATCase catalyzes the first reaction of the pyrimidine nucleotide biosynthesis pathway (CTP and UTP) and uses Asp as a substrate. The structure of ATCase consists of catalytic and regulatory subunits. The ATCase has two conformational states R and T. The symmetry between these two states is conserved.

The kinetics of ATCase (the initial rate of ATCase with different concentrations of aspartate) is characterized by a sigmoid curve. This indicates that ATCasa has a cooperative behavior.

ATCase is feedback inhibited by CTP. The sigmoid curve of ATCase, in the presence of CTP, is to the right of the sigmoid curve of ATCase in the absence of CTP. An increase in the value of the Michaelis-Menten constant (K _m) is evidenced.

That is, in the presence of CTP, ATCase requires a higher concentration of aspartate to reach half the maximum rate (V _max), compared to ATCase in the absence of CTP.

In conclusion, CTP is a heterotropic negative allosteric effector because it decreases the affinity of ATCase for aspartate. This behavior is known as negative cooperativity.

PFK – 1

PFK-1 catalyzes the third reaction of the glycolysis pathway. This reaction consists of the transfer of a phosphate group from ATP to fructose 6-phosphate. The structure of PFK-1 is a tetramer, which exhibits two conformational states R and T. The symmetry between these two states is conserved.

The kinetics of PFK-1 (the initial rate with different concentrations of fructose 6-phosphate) exhibits a sigmoid curve. PFK-1 is subject to complex allosteric regulation by ATP, AMP and frutose-2,6-bisphosphate, namely:

The sigmoid curve of PFK-1, in the presence of a high concentration of ATP, is to the right of the sigmoid curve at a low concentration of ATP (Figure 4). An increase in the value of the Michaelis-Menten constant (K _m) is evidenced.

In the presence of a high concentration of ATP, PFK-1 requires a higher concentration of fructose 6-phosphate to reach half the maximum rate (V _max).

In conclusion, ATP, in addition to being a substrate, is a negative heterotropic allosteric effector because it decreases the affinity of PFK-1 for fructose 6-phosphate.

The sigmoid curve of PFK-1, in the presence of AMP, lies to the left of the sigmoid curve of PFK-1 in the presence of ATP. That is, AMP eliminates the inhibitory effect of ATP.

In the presence of AMP, PFK-1 requires a lower concentration of fructose 6-phosphate to reach half the maximum rate (V _max). This is manifested in the fact that there is a decrease in the value of the Michaelis-Menten constant (K _m).

In conclusion, AMP is a positive heterotropic allosteric effector because it increases the binding affinity of PFK-1 for fructose 6-phosphate. Frutose-2,6-bisphosphate (F2,6BP) is a potent allosteric activator of PFK-1 (Figure 5), and its behavior is similar to that of AMP.

The MWC model is common, but not universal

Of the total protein structures deposited in PDB (Protein data bank), half are oligomers and the other half are monomers. It has been shown that cooperativity does not require multiple ligands, or the assembly of multiple subunits. This is the case for glucokinase and other enzymes.

Glucokinase is monomeric, has a polypeptide chain, and exhibits sigmoidal kinetics in response to increased blood glucose concentration (Porter and Miller, 2012; Kamata et al., 2004).

There are different models that explain cooperative kinetics in monomeric enzymes, namely: mnemonic model, ligand-induced slow transition model, random addition of substrates in biomolecular reactions, types of slow conformational changes, among others.

Studies of the structure of glucokinase have supported the mnemonic model

Normal human glucokinase has a K _m of 8 mM for glucose. This value is close to the blood glucose concentration.

There are patients who suffer from pes-resistant hyperinsulinemia of childhood (PHHI). Glucokinase in these patients has a lower K _m for glucose than normal glucokinases, and cooperativity is significantly reduced.

Consequently, these patients possess a glucokinase variant that is hyperactive, which in severe cases can be fatal.

Applications of allosterism

Allostry and catalysis are closely linked. Because of this, allosteric effects can affect catalysis characteristics such as ligand binding, ligand release.

Allosteric binding sites may be targets for new drugs. This is because the allosteric effector can influence the function of the enzyme. Identification of allosteric sites is the first step in the discovery of drugs that enhance enzyme function.

References

1. Changeux, JP 2012. Allostery and the Monod-Wyman-Changeux model After 50 years. Annual Review of Biophysics and Biomolecular Structure, 41: 103–133.
2. Changeux, JP 2013. 50 years of allosteric interactions: the twists and turns of the models. Molecular Cell Biology, in Nature Reviews, 14: 1–11.
3. Goodey, NM and Benkovic, SJ 2008. Allosteric regulation and catalysis emerge via a common route. Nature Chemical Biology, 4: 274-482.
4. Kamata, K., Mitsuya, M., Nishimura, T., Eiki, Jun-ichi, Nagata, Y. 2004. Structural basis for allosteric regulation of the monomeric allosteric enzyme human glucokinase. Structure, 12: 429–438.
5. Koshland, DE Jr., Nemethy, G., Filmer, D. 1966. Comparison of experimental binding data and theoretical models in proteins containing subunits. Biochemistry, 5: 365-385.
6. Monod, J., Wyman, J., Changeux, JP 1965. On the nature of allosteric transitions: a plausible model. Journal of Molecular Biology, 12: 88–118.
7. Nelson, DL and Cox, MM, 2008. Lehninger – Principles of Biochemistry. WH Freeman and Company, New York.
8. Porter, CM and Miller, BG 2012. Cooperativity in monomeric enzymes with single ligand-binding sites. Bioorganic Chemistry, 43: 44-50.
9. Voet, D. and Voet, J. 2004. Biochemistry. John Wiley and Sons, USA.