BIOCHEMISTRY - Textbook - Ostapchenko L. I. - 2012

Chapter 5. STRUCTURE, PROPERTIES, AND FUNCTIONS OF PROTEINS

5.4. Protein Functioning

5.4.1. The Active Site of Proteins and the Selectivity of Ligand Binding

The Active Site of a protein is a specific region of the protein molecule located in its cleft (or "pocket"), formed by amino acid side chains, and capable of binding to a Ligand in a complementary manner. In the linear sequence of the polypeptide chain, the residues that form the active site may be located at a considerable distance from one another.

The high Specificity of protein-ligand binding is ensured by the complementarity between The Structure of the protein's active site and that of the ligand (Fig. 5.25).

Complementarity refers to the spatial and chemical correspondence between interacting molecules. A ligand must be able to enter and spatially match the conformation of the active site. This match may be partial, but due to the conformational flexibility of the protein, the active site can undergo minor adjustments and "fit" the ligand. Furthermore, bonds must form between the Functional groups of the ligand and The amino acid side chains that make up the active site, holding the ligand in place. The bonds between the ligand and the protein's active site can be either non-covalent (ionic, hydrogen, hydrophobic) or covalent.

The active site of a protein is a region of the protein molecule relatively isolated from the external environment and formed by amino acid residues. Within this region, each residue, owing to

its individual size and functional groups, shapes the "topography" of the active site. The combination of such Amino Acids into a single functional complex alters the reactivity of their side chains. The amino acid residues that comprise the active site are often referred to as an amino acid "ensemble".

Class="center">

Fig. 5.25. Protein-ligand interaction:

A and B - non-complementary interaction and disruption of bonds between the protein and the ligand; C - complementary interaction of the protein with the ligand

The unique Properties of the active site depend not only on The chemical properties of the amino acids that form it, but also on their precise spatial orientation relative to one another. Therefore, even minor disruptions in the overall protein conformation resulting from point Mutations in its Primary Structure or changes in environmental conditions can alter the chemical and Functional Properties of the side chains forming the active site, impairing protein-ligand binding and its biological function. During Denaturation, the active site is destroyed, leading to a loss of biological activity.

Often, the active site is formed in such a way that access of Water to the functional groups of its side chains is restricted, thereby creating optimal conditions for ligand binding to the amino acid residues.

In some cases, a ligand attaches to only a specific reactive atom, such as the binding of _О2 to the iron in Myoglobin or Hemoglobin. However, the ability of this atom to selectively interact with O₂ is determined by The properties of the side chains surrounding the iron atom within the heme group. Heme is also found in other Proteins, such as Cytochromes. Yet, the function of the iron atom in cytochromes is different; it acts as an electron transfer mediator between substances, shifting between ferrous (iron(II)) and ferric (iron(III)) states.

The ligand-binding center of a protein is frequently located between domains. For instance, the proteolytic enzyme Trypsin, which is involved in the Hydrolysis of peptide bonds in dietary proteins within the intestine, features two domains separated by a cleft. The inner surface of this cleft is formed by amino acid side chains from these domains that are located far apart in the polypeptide chain (Ser₁₇₇, His₄₀, Asp₈₅).

Different domains within a protein can move relative to one another during ligand interaction, facilitating subsequent protein function. A classic example is hexokinase, an enzyme that catalyzes The transfer of a phosphoryl group from ATP to a glucose molecule (during its phosphorylation). The active site of hexokinase is located in the cleft between two domains (Fig. 5.26). Upon glucose binding, the surrounding domains close in, trapping the substrate and thereby facilitating its subsequent phosphorylation.

Fig. 5.26. Binding of hexokinase to glucose

A fundamental property underlying Protein Functions is the selectivity of binding specific ligands to particular Regions of the protein molecule. These ligands can be inorganic (frequently Metal Ions) or organic substances, as well as low-molecular-weight or high-molecular-weight compounds. Some ligands undergo chemical modification upon binding to the active site (such as substrates reacting within an enzyme's active site). Others bind only transiently during the functional phase (e.g., _О2 transported by hemoglobin), while some remain permanently bound and play an auxiliary role in protein function (e.g., the iron incorporated into hemoglobin).

In cases where the amino acid residues forming the active site cannot single-handedly ensure the protein's function, non-protein molecules may attach to specific regions of the active site. For instance, the active site of many Enzymes contains a metal ion (cofactor) or a non-protein organic molecule (coenzyme). A non-protein moiety that is tightly bound to the active site and essential for its function is called a prosthetic group. Myoglobin, hemoglobin, and cytochromes all contain an iron-bearing prosthetic group known as heme within their active sites.

The association of protomers in an oligomeric protein is an example of high-molecular-weight ligand interaction. Each protomer associated with other protomers serves as a ligand for them, just as they do for it.

Sometimes, the binding of a ligand alters the protein's conformation, which in turn creates a binding site for other ligands. For example, upon binding four Ca²⁺ ions at specific sites, the protein calmodulin acquires The ability to interact with various enzymes, thereby modulating their activity.

The Rate of protein-ligand interaction is determined by the concentrations of both the protein and the ligand in solution, as well as the degree of complementarity between them.

The dissociation constant is a measure of the active site's affinity for a ligand. Because Protein-ligand interactions are reversible, the process can be described by the following equation:

where P is the protein, L is the ligand, PL is the protein-ligand complex, K₁ is the association rate constant of the protein-ligand binding, and K_-1 is the dissociation rate constant of the PL complex.

When the rates of formation and dissociation of the complex are equal, the system reaches a state of equilibrium:

Hence,

The ratio of the dissociation rate constant of the [PL] complex to its formation rate constant is called the dissociation constant (K_d) of the [PL] complex. The smaller the K_d, the more ligand molecules are bound to the protein, the higher the complementarity between P and L, and the greater the ligand's affinity for the protein. In other words, there is an inverse relationship between K_d and ligand-protein affinity.

Sometimes, when describing the protein-ligand binding process, the reciprocal of K_d is used, which is called the binding constant (K_b) or association constant:

There is a direct proportional relationship between K_b and ligand-protein affinity. At a constant protein concentration, increasing the ligand concentration leads to an increase in the [PL] concentration. This dependence takes the form of a hyperbolic curve (Fig. 5.27). The curve approaches a maximum when, at a certain ligand concentration, all protein molecules exist in the ligand-bound state (protein saturation with the ligand occurs). The degree of protein saturation with the ligand can be expressed by the equation:

(where P₀ is the initial protein concentration before The addition of the ligand).

Fig. 5.27. Protein-ligand saturation curve

At half-saturation of the protein with the ligand, the concentrations of [PL] and [P] are equal. It follows from the K_d equation given above that K_d = [L], meaning that K_d is numerically equal to the ligand concentration at which 50% of the protein is complexed with the ligand. Therefore, the saturation curve can be used to determine K_d and evaluate the affinity of a given protein for the ligand.

As described above, with increasing ligand concentration, protein saturation is limited by its own concentration. In the presence of excess ligand, all protein molecules are incorporated into the [PL] complex. However, if the protein concentration is increased, The amount of [PL] begins to increase in proportion to the protein amount. The concentration of the [PL] complex can be monitored by measuring Light absorption. Given that its amount is proportional to the protein concentration, the protein concentration in solution can be determined using the plotted graph (Fig. 5.28).

Fig. 5.28. Graph of changes in light absorption reflecting the concentration of the [PL] complex as a function of protein P concentration:

the A-axis records the change, e.g., in light absorption caused by The formation of the [PL] complex