An active site is the part of an enzyme that directly binds to a substrate and carries a reaction. It contains catalytic groups which are amino acids that promote formation and degradation of bonds. By forming and breaking these bonds, enzyme and substrate interaction promotes the formation of the transition state structure. Enzymes help a reaction by stabilizing the transition state intermediate. This is accomplished by lowering the energy barrier or activation energy- the energy that is required to promote the formation of transition state intermediate. The three dimensional cleft is formed by the groups that come from different part of the amino acid sequences. The active site is only a small part of the total enzyme volume. It enhances the enzyme to bind to substrate and catalysis by many different weak interactions because of its nonpolar microenvironment. The weak interactions includes the Van der Waals, hydrogen bonding, and electrostatic interactions. The arrangement of atoms in the active site is crucial for binding spectificity. The overall result is the acceleration of the reaction process and increasing the rate of reaction. Furthermore, not only do enzymes contain catalytic abilities, but the active site also carries the recognition of substrate.
The enzyme active site is the binding site for catalytic and inhibition reactions of enzyme and substrate; structure of active site and its chemical characteristic are of specific for the binding of a particular substrate. The binding of the substrate to the enzyme causes changes in the chemical bonds of the substrate and causes the reactions that lead to the formation of products. The products are released from the enzyme surface to regenerate the enzyme for another reaction cycle.
The active site is in the shape of a three-dimensional cleft that is composed of amino acids from different residues of the primary amino acid sequence. The amino acids that play a significant role in the binding specificity of the active site are usually not adjacent to each other in the primary structure, but form the active site as a result of folding in creating the tertiary structure. This active site region is relatively small compared to the rest of the enzyme. Similar to a ligand-binding site, the majority of an enzyme (non-binding amino acid residues) exist primarily to serve as a framework to support the structure of the active site by providing correct orientation. The unique amino acids contained in an active site promote specific interactions that are necessary for proper binding and resulting catalysis. Enzyme specificity depends on the arrangement of atoms in the active site. Complementary shapes between enzyme and substrate(s) allow a greater amount of weak non-covalent interactions including electrostatic forces, Van der Waals forces, hydrogen bonding, and hydrophobic interactions. Specific amino acids also allow the formation of hydrogen bonds. That shows the uniqueness of the microenvironment for the active site.
To locate the active site, the enzyme of interest is crystallized in the presence of an analog. The analog’s resemblance of the original substrate would be considered a potent competitive inhibitor that blocks the original substrates from binding to the active sites. One can then locate the active sites on an enzyme by following where the analog binds.
Active Site vs. Regulatory Site
An enzyme, for example ATCase, contains two distinct subunits: an active site and a regulatory site. The active site is the catalytic subunit, whereas the regulatory site has no catalytic activity. The two subunits on the enzyme was confirmed by John Gerhart and Howard Schachman by doing the ultracentrifugation experiment. First, they treated the ATCase with p-hydroxymercuribenzoate to react with the sulfhydryl groups and dissociate the two subunits. Because the two subunits differ in sizes with the catalytic subunit being larger, results of centrifuging the dissociated subunits showed two sedimentations compared to the one sediment of the native enzyme. This proved that ATCase, like many other enzymes, contain two sites for substrates to bind.
There are three different models that represent enzyme-substrate binding: the lock-and-key model, the induced fit model, and transition-state model.
The lock-and-key model was proposed by Emil Fischer in 1890. This model presumes that there is a perfect fit between the substrate and the active site—the two molecules are complementary in shape. Lock-and-key is the model such that active site of enzyme is good fit for substrate that does not require change of structure of enzyme after enzyme binds substrate
The induced-fit model involves the changing of the conformation of the active site to fit the substrate after binding. Also, in the induced-fit model, it was stated that there are amino acids that aid the correct substrate to bind to the active site which leads to shaping of the active site to the complementary shape. Induced fit is the model such that structure of active site of enzyme can be easily changed after binding of enzyme and substrate.
The binding in the active site involves hydrogen bonding, hydrophobic interactions and temporary covalent bonds. The active site will then stabilize the transition state intermediate to decrease the activation energy. But the intermediate is most likely unstable, allowing the enzyme to release the substrate and return to the unbound state.
The transition-state model starts with an enzyme that binds to a substrate. It requires energy to change the shape of substrate. Once the shape is changed, the substrate is unbound to the enzyme, which ultimately changes the shape of the enzyme. An important aspect of this model is that it increases the amount of free energy.
A binding site is a position on a protein that binds to an incoming molecule that is smaller in size comparatively, called ligand.
In proteins, binding sites are small pockets on the tertiary structure where ligands bind to it using weak forces (non-covalent bonding). Only a few residues actually participate in binding the ligand while the other residues in the protein act as a framework to provide correct conformation and orientation. Most binding sites are concave, but convex and flat shapes are also found.
A ligand-binding site is a place of the mass chemical specificity and affinity on protein that binds or forms chemical bonds with other molecules and ions or protein ligands. The affinity of the binding of a protein and a ligand is a chemically attractive force between the protein and ligand. As such, there can be competition between different ligands for the same binding site of proteins, and the chemical reaction will result in an equilibrium state between bonding and non-bonding ligands. The saturation of the binding site is defined as the total number of binding sites that are occupied by ligands per unit time.
The most common model of enzymatic binding sites is the induced fit model. It differs from the more simple "Lock & key" school of thought because the induced fit model states that the substrate of an enzyme does not fit perfectly into the binding site. With the "lock & key" model it is assumed that the substrate is a relatively static model that does not change its conformation and simply binds to the active site perfectly. According to the induced fit model, the binding site of an enzyme is complementary to the transition state of the substrate in question, not the normal substrate state. The enzyme stabilizes this transition state by having its NH3+ residues stabilize the negative charge of the transition state substrate. This results in a dramatic decrease in the activation energy required to bring forth the intended reaction. The substrate is then converted to its product(s) by having the reaction go to equilibrium quicker.
Properties that Affect Binding
- Complementarity:Molecular recognition depends on the tertiary structure of the enzyme which creates unique microenvironments in the active/binding sites. These specialized microenvironments contribute to binding site catalysis.
- Flexibility:Tertiary structure allows proteins to adapt to their ligands (induced fit) and is essential for the vast diversity of biochemical functions (degrees of flexibility varies by function)
- Surfaces:Binding sites can be concave, convex, or flat. For small ligands – clefts, pockets, or cavities. Catalytic sites are often at domain and subunit interfaces.
- Non-Covalent Forces:Non-covalent forces are also characteristic properties of binding sites. Such characteristics are: higher than average amounts of exposed hydrophobic surface, (small molecules – partly concave and hydrophobic), and displacement of water can drive binding events.
- Affinity: Binding ability of the enzyme to the substrate (can be graphed as partial pressure increases of the substrate against the affinity increases (0 to 1.0); affinity of binding of protein and ligand is chemical attractive force between the protein and ligand.
Enzyme inhibitors are molecules or compounds that bind to enzymes and result in a decrease in their activity. An inhibitor can bind to an enzyme and stop a substrate from entering the enzyme's active site and/or prevent the enzyme from catalyzing a chemical reaction. There are two categories of inhibitors.
- 'Irreversible Inhibitors[non competitive only]
- Reversible Inhibitors[both competitive and non competitive]
Inhibitors can also be present naturally and can be involved in metabolism regulation. For example. negative feedback caused by inhibitors can help maintain homeostasis in a cell. Other cellular enzyme inhibitors include proteins that specifically bind to and inhibit an enzyme target. This is useful in eliminating harmful enzymes such as proteases and nucleases.
Examples of inhibitors include poisons and many different types of drugs. and also heavy metals such as lead and cyanide
Irreversible inhibitors covalently bind to an enzyme, cause chemical changes to the active sites of enzymes, and cannot be reversed. A main role of irreversible inhibitors include modifying key amino acid residues needed for enzymatic activity. They often contain reactive functional groups such as aldehydes, alkenes, or phenyl sulphonates. These electrophilic groups are able to react with amino acid side chains to form covalent products. The amino acid components are residues containing nucleophilic side chains such as hydroxyl or sulphydryl groups such as amino acids serine, cysteine, threonine, or tyrosine.
First, irreversible inhibitors form a reversible non-covalent complex with the enzyme (EI or ESI). Then, this complex reacts to produce the covalently modified irreversible comple EI*. The rate at which EI* is formed is called the inactivation rate or kinact. Binding of irreversible inhibitors can be prevented by competition with either substrate or a second, reversible inhibitor since formation of EI may compete with ES.
In addition, some reversible inhibitors can form irreversible products by binding so tightly to their target enzyme. These tightly-binding inhibitors show kinetics similar to covalent irreversible inhibitors. As shown in the figure, these inhibitors rapidly bind to the enzyme in a low-affinity EI complex and then undergoes a slower rearrangement to a very tightly bound EI* complex. This kinetic behavior is called slow-binding. Slow-binding often involves a conformational change as the enzyme "clamps down" around the inhibitor molecule. Some examples of these slow-binding inhibitors include important drugs such as methotrexate and allopurinol.
Reversible inhibitors bind non-covalently to enzymes, and many different types of inhibition can occur depending on what the inhibitors bind to. The non-covalent interactions between the inhibitors and enzymes include hydrogen bonds, hydrophobic interactions, and ionic bonds. Many of these weak bonds combine to produce strong and specific binding. In contrast to substrates and irreversible inhibitors, reversible inhibitors generally do not undergo chemical reactions when bound to the enzyme and can be easily removed by dilution or dialysis.
There are three kinds of <cap>reversible inhibitors</cap>: competitive, noncompetitive, and uncompetitive/mixed inhibitors.
- Competitive inhibitors, as the name suggests, compete with substrates to bind to the enzyme at the same time. The inhibitor has an affinity for the active site of an enzyme where the substrate also binds to. This type of inhibition can be overcome by increasing the concentrations of substrate, out-competing the inhibitor. Competitive inhibitors are often similar in structure to the real substrate.
Competitive inhibitor binds to active site of enzyme and decreases amount of binding of substrate or ligand to enzyme, such that Km is increased and Vmax not changed. The chemical reaction can be reversed by increasing concentration of substrate.
- Uncompetitive inhibitors bind to the enzyme at the same time as the enzyme's substrate. However, the binding of the inhibitor affects the binding of the substrate, and vice-versa. This type of inhibition cannot be overcome, but can be reduced by increasing the concentrations of substrate. The inhibitor usually follows an allosteric effect where it binds to a different site on the enzyme than the substrate. This binding to an allosteric site changes the conformation of the enzyme so that the affinity of the substrate for the active site is reduced.
Uncompetitive inhibitor binds to enzyme-substrate complex to stops enzyme from reacting with substrate to form product, as such, it works well at higher substrate and enzyme concentrations that substrates are bonded to enzymes; the binding results in decreasing concentration of substrate binding to enzyme, Km, and Vmax, and increasing binding affinity of enzyme to substrate.
- Non-competitive inhibitors bind to the active site and reduces the activity but does not affect the binding of the substrate. Therefore, the extent of inhibition depends on the concentration of the substrate.
Noncompetitive inhibitor binds to other site that is not active site of enzyme that changes structure of enzyme; therefore, blocks enzyme binding to substrate that stops enzyme activity and decreases rate of chemical reaction of enzyme and substrate, which can not be changed by increasing concentration of substrate; the binding decreases Vmax and not changes Km of the chemical reaction.
Quantitative Description of Reversible InhibitorsEdit
Most reversible inhibitors follow the classic Michaelis-Menten scheme, where an enzyme (E) binds to its substrate(S) to form an enzyme-substrate complex (ES). km is the Michaelis constant that corresponds to the concentration of the substrate when the velocity is half the maximum. Vmax is the maximum velocity of the enzyme.
- Competitive inhibitors can only bind to E and not to ES. They increase Km by interfering with the binding of the substrate, but they do not affect Vmax because the inhibitor does not change the catalysis in ES because it cannot bind to ES.
- Uncompetitive inhibitors are able to bind to both E and ES, but their affinities for these two forms of the enzyme are different. Therefore, these inhibitors increase Km and decrease Vmax because they interfere with substrate binding and hamper catalysis in the ES complex.
- Non-competitive inhibitors have identical affinities for E and ES. They do not change Km, but decreases Vmax.