Structural Biochemistry/Enzyme Catalytic Mechanism/Binding

The protein function of binding is very specific. The ability of binding is dependent on the tertiary structure of the protein, also known as the three-dimensional structure of the protein. The area of the protein that is bound to another molecule, such as a ligand, is called the binding site. The binding site is often a crevice on the surface of the protein. The molecule that binds to the protein changes the chemical conformation of the protein.

•The active site is also the site of inhibition of enzymes

•The active site of an enzyme contains the catalytic and binding sites

•The structure and chemical properties of the active site allow the recognition and binding of the substrate

•Protein functions such as molecular recognition and catalysis depend on complementarity

•Molecular recognition depends on specialized microenvironments that result from protein tertiary structure

•Specialized microenvironments at binding sites contribute to catalysis

An example of binding with a protein is the ligand-binding protein of hemoglobin, which transports oxygen from the lungs to other important organs and tissues within humans.

Protein properties that affect binding

Recognition/Specificity

An enzyme is specific to the substrate it binds to. It is dependent on structure and placement as well as the sequence of amino acids of the substrate. If the sequence is complementary then it ensures the binding of the two components. In the case of proteases, the residues that are not responsible for catalysis may be responsible for the recognition and alignment of the molecule, setting it up so that it can have one of its bonds hydrolyzed by the enzyme.

Proteases in particular tend to recognize the side chains of the amino acid it intends to cleave. Oftentimes it is the carbonyl carbon - amide group that succeeds the recognized side chain.

Note: image is the protease of an HIV-1

It is possible to gauge whether an amino residue plays a role in the enzymatic activity of a protease by inducing site-directed mutagenesis to the amino acid in question. If the amino acid in question does play a role in specificity or catalysis, post-mutation the enzyme will have a decrease in its enzymatic activity.

Complementarity

The concept of complementarity can be understood in terms of the lock and key model of protein binding (as shown in the figure below). Essentially, the surface of the protein involved in binding exhibits a shape that is complementary to the binding ligand. This allows for protein recognition, binding specificity and affinity. Protein binding can be further explained by the induced fit model. In this model, the protein's binding site also exhibits complementarity, but to a lesser degree than the lock and key model. Once, the ligand is already at the binding site, the protein can adjust the shape of its binding site to better fit and bind the ligand. This concept of protein flexibility is explained in more details in the next section.

Surface

Binding usually occurs at the surface of the protein. See Nature of Binding Sites (below) for more detailed information. The image below shows an example of how a protein's surface recognizes molecules. This is the binding between a sex hormone and globulin.

Non-Covalent Forces

Ligand to protein binding typically occurs through non-covalent forces. The weak interactions of non-covalent forces allow for easy exchange between molecules. For instance, hemoglobin traps oxygen in its binding site at the lungs and releases it to the tissues. It is able to perform the task very efficiently because the binding is strong enough to tightly hold on to the oxygen when it is in the oxygen-saturated lungs, yet weak enough to allow for an easy release of oxygen to the oxygen-deficient tissues.

Flexibility

The concept of flexibility can be demonstrated through the idea of induced fit. An enzyme that has a site that originally is not complementary to a substrate may become complementary upon the binding of the substrate. The site of binding changes to a shape that accepts the substrate. This indicates that the enzyme is indeed flexible and the conformation can be changed by the influence of the substrate.

The induced fit model asserts that the binding site of an enzyme is optimized for the transition state of the substrate, not the normal state. This is so that it can easily stabilize the transition state once it is bound to the enzyme, thereby decreasing the activation energy of the reaction and bringing the reaction to equilibrium much quicker .

Nature of Binding sites

The binding sites of enzymes has several common features. The active site is a site formed by amino acids that are connected to different parts of the protein. The specific positioning of the amino acids work together to form a three dimensional cleft or crevice. The active site of an enzyme takes up a very small portion of the total volume of the enzyme. The amino acids that do not play a role in enzymatic activity are there to make up for the structure. The many amino acids form a three dimensional structure of the molecule that allows the interaction of the active sites to work with each other and to have reactions with other molecules. Active sites also possess unique micro environments. These unique environments may contain polar or nonpolar residues that each have their own ways for interacting with nearby substrates. In addition, substrates are bound to enzymes by many weak forces that include van der Waals forces, hydrogen bonding, and hydrophobic interactions. Although these forces may be weak individually, the large number of forces acting together contributes to the stability of the binding site of the enzyme-substrate complex. Finally, the specific binding sites of an enzyme depend on the specific arrangement of the molecule. Here is an example where one structure leads to an specific function.

Binding sites for large ligands can be either flat, convex, or concave while binding sites for small ligands tend to be exclusively concave. One characteristic of binding sites is that it contain a significantly greater amount of exposed hydrophobic surfaces than other parts. In addition, it is also important for binding sites to possess the ability to bound to ligands firmly, but not so firmly that it will be hard for the ligand to be released. Therefore, weaker non-covalent interaction is characteristic of binding sites because it allows for an easy exchange. This concept is particularly important for hemoglobin because its major function is to transport oxygen from the lungs to the tissues. In order to efficiently do its job, its binding affinity to oxygen must be sufficiently strong, but at the same time, not too strong otherwise the hemoglobin would not be able to release the bound oxygen once it's in the tissue. Aside from binding affinity, binding specificity is also very important. Specialized and specific micro environments at binding sites are necessary for efficient binding. Lastly, the displacement of water is often typical for binding sites because it is able to promote the binding process.

How Enzyme Catalytic Mechanism/Binding relates to Pharmaceutical field

Pharmaceutical drugs work the same way Enzymes work. They bind to a specific binding site that either inhibits or activates a specific biological action. Caffeine for example is a really known molecule that a lot of people use every day, by drinking coffee or tea, to keep them awake. The reason behind the relationship between Caffeine and alertness and wakefulness is the big structural similarity between the caffeine molecule, adenosine and cyclic adenosine phosphate. The structures similarity allows caffeine molecules to bind to the same binding site of receptors or enzymes that reacts with adenosine derivatives. Adenosine has a very important role in the regulation of brain activity. The human brain builds up adenosine molecules during the day. When the level of built up adenosine increases in the human brain, adenosine starts binding to its binding sites (receptors) in the human brain which activates mechanisms that lead to drowsiness and sleep. Since caffeine has the same structural molecule it binds to the same receptors that adenosine binds to, preventing adenosine to bind to that specific receptor in the human brain and delaying the sleeping and drowsiness process.

Pharmaceutical drugs have pretty much the same role. They bind to a protein, enzyme, or a receptor that prevents binding of a specific molecule in the human body to bind to that specific receptor and therefore they inhibit a specific mechanism. A specific drug or molecule can bind to more than one binding site as mentioned earlier in the caffeine case. That means that the same drug can inhibit more than one biological mechanism at the same time by binding to two different binding sites. Binding to more than one binding site can explain the side effects of a specific prescribed drug. Furthermore, pharmaceutical and drug development scientists are currently trying to correlate and create a large network of the current drugs with their side effects, binding sites, structures, and role. The reason there is so much research is done in that field is that scientists strongly believe that discovering drugs that are already there that can cure some other diseases has a very high probability because of the presence of a wide range of drugs. Spending enough money on such projects can save a huge amount of money spent on drugs discovery. Discovering a drug can cost $800 million that is not including the huge amount of time and hassle it takes to test out the drug and then goes in market. Tamoxifen for instance has thirty six discovered binding sites in the human body. Tamoxifen’s molecule has structural similarity to estrogen but it is missing a part that activates breast cancer cells production. Tamoxifen’s secondary binding sites suggest that the drug can be used to cure some other diseases. Through bioinformatics, researchers are working hard developing websites and databases that helps ease drug discovery process and makes it more effective and less costly. Promisuous([1]) database is a tool that has a lot of information about drugs, their structures, binding sites (targets) and metabolic pathways that a specific drug goes through. The tool is really helpful and it has all the needed information that were collected through other important websites and databases like PubChem, Protein Data Base, and Uniprot. It a great tool that helps researchers find more information about drugs in just one place faster and eases drug discovery process in the future.

References

Jeremy Berg, Biochemistry 6ed.