Action and Nature of Enzymes

Enzymes are the biocatalysts with high molecular weight proteinous compound. It enhances the reactions which occur in the body during various life processes. It helps the substrate by providing the surface for the reaction to occur. The enzyme comprises hollow spaces occupying groups such as -SH, -COOH, and others on the outer surface. The substrate which has an opposite charge of the enzyme fits into these spaces just like a key fits into a lock. This substrate binding site is called the active site of an enzyme (E).

The favourable model of enzyme-substrate interaction is called the induced-fit model. This model states that the interaction between substrate and enzyme is weak, and these weak interactions induce conformational changes rapidly and strengthen binding and bring catalytic sites close enough to substrate bonds.

There are four possible major mechanisms of catalysis:

Catalysis by Bond Strain

The induced structural rearrangements in this type of catalysis produce strained substrate bonds that attain transition state more easily. The new conformation forces substrate atoms and catalytic groups like aspartate into conformations that strain substrate bonds.

Covalent Catalysis

The substrate is oriented to active place on the enzymes in such a manner that a covalent intermediate develops between the enzyme and the substrate, in catalysis that occurs by covalent mechanisms. The best example of this involves proteolysis by serine proteases that have both digestive enzymes and various enzymes of the blood clotting cascade. These proteases have an active site serine whose R group hydroxyl produces a covalent bond with a carbonyl carbon of a peptide bond and causes hydrolysis of the peptide bond.

Catalysis Involving Acids and Bases

Other mechanisms also contribute to the completion of catalytic events that are initiated by strain mechanism like the use of glutamate as a general acid catalyst.

Catalysis by Orientation and Proximity

Enzyme-substrate interactions bring reactive groups into proximity with one another. Also, groups like aspartate are chemically reactive, and their proximity and towards the substrate favours their involvement in catalysis.

Action and Nature of Enzymes

Once substrate (S) binds to this active site, they form a complex (intermediate-ES) which then produces the product (P) and the enzyme (E) The substrate which gets attached to the enzyme has a specific structure, and that can only fit in a particular enzyme. Hence by providing a surface for the substrate, an enzyme slows down the activation energy of the reaction. The intermediate state where the substrate binds to the enzyme is called the transition state. By breaking and making the bonds, the substrate binds to the enzyme (remains unchanged), which converts into the product and later splits into product and enzyme. The free enzymes then bind to other substrates, and the catalytic cycle continues until the reaction completes.

The enzyme action basically happens in two steps:

Step1: Combining of enzyme and the reactant/substrate.

E+S → [ES]

 Step 2: Disintegration of the complex molecule to give the product.

[ES]→E+P

Thus, the whole catalyst action of enzymes is summarized as:

E + S → [ES] → [EP] → E + P

Biological Catalysts

Catalysts are the substances which play a significant role in the chemical reaction. Catalysis is the phenomenon by which the rate of a chemical reaction is altered/ enhanced without changing themselves. During a chemical reaction, a catalyst remains unchanged, both in terms of quantity and chemical properties. An enzyme is one such catalyst which is commonly known as the biological catalyst.  Enzymes present in the living organisms enhance the rate of reactions which take place within the body.

Enzymes, the biological catalysts are highly specific, catalyzing a single chemical reaction or a very few closely related reactions. The exact structure of an enzyme and its active site determines the specificity of the enzyme. Substrate molecules bind themselves at the enzyme’s active site. Substrates initially bind to the enzymes by noncovalent interactions, including hydrogen bonds, ionic bonds, and hydrophobic interactions. Enzymes lower the activation energy and the reactions proceed toward equilibrium more rapidly than the uncatalyzed reactions. Both prokaryotic and eukaryotic cells commonly use allosteric regulation in responding to changes in conditions within the cells.

The nature of enzyme action and factors affecting the enzyme activity are discussed below.

Factors Affecting Enzyme Activity

The conditions of the reaction have a great impact on the activity of the enzymes. Enzymes are particular about the optimum conditions provided for the reactions such as temperature, pH, alteration in substrate concentration, etc.

Generally, an increase in temperature increases the activity of enzymes. Because enzymes function in cells, the optimum conditions for most enzymes are moderate temperatures. At elevated temperatures, at a certain point activity decreases dramatically when enzymes are denatured. Purified enzymes in diluted solutions are denatured more rapidly than enzymes in crude extracts. Incubation of enzymes for long periods may also denature enzymes. It is more suitable to use a short incubation time in order to measure the initial velocities of the enzyme reactions.

The International Union of Biochemistry recommends 30 °C as the standard assay temperature. Most enzymes are very sensitive to changes in pH. Only a few enzymes function optimally below pH 5 and above pH 9. The majority of enzymes have their pH-optimum close to neutrality. The change in pH will change the ionic state of amino acid residues in the active site and in the whole protein. The change in the ionic state may change substrate binding and catalysis. The choice of substrate concentration is also crucial because, at low concentrations, the rate is dependent on the concentration, but at high concentrations, the rate is independent of any further increase in substrate concentration.

Active site

Enzymatic catalysis relies on the action of amino acid side chains arrayed in the active centre. Enzymes bind the substrate into a region of the active site in an intermediate conformation.

The active site is often a pocket or a cleft formed by the amino acids that participate in substrate binding and catalysis. The amino acids that make up the active site of an enzyme are not contiguous to one another along the primary amino acid sequence. The active site amino acids are brought to the cluster in the right conformation by the 3-dimensional folding of the primary amino acid sequence. Of the 20 different amino acids that make up protein, the polar amino acids, aspartate, glutamate, cysteine, Serine, histidine, and lysine have been shown most frequently to be active site amino acid residues. Usually, only two to three essential amino acid residues are directly involved in the bond leading to product formation. Aspartate, glutamate, and histidine are the amino acid residues that also serve as proton donors or acceptors.

Temperature and pH

Enzyme as a biological substance acts within temperature limits.  This s very important in enzymology because outside their temperature limits enzyme may become inactive or slow down in action. Enzymes have an optimum temperature and pH where they work best. Changes in pH can alter critical ionization states, while changes in temperature can disrupt important bonds. Deviations from these optimum conditions will disrupt the enzyme’s structure and impact on its kinetics, eventually completely denaturing and disabling it. pH is also very important because these are physical factors that determine biological activities. It is noteworthy that action of salivary amylase stops when the chewed food gets to the stomach due to increased pH of the environment.  Enzymes require an optimum temperature and pH for their action. The temperature or pH at which a compound shows its maximum activity is called optimum temperature or optimum pH, respectively. As mentioned earlier, enzymes are protein compounds. A temperature or pH more than optimum may alter the molecular structure of the enzymes. Generally, an optimum pH for enzymes is considered to be ranging between 5 and 7.

• Optimum T°
• The greatest number of molecular collisions
• human enzymes = 35°- 40°C
• body temp = 37°C
• Heat: increase beyond optimum T°
• The increased energy level of molecules disrupts bonds in enzyme & between enzyme & substrate H, ionic = weak bonds
• Denaturation = lose 3D shape (3° structure)
• Cold: decrease T°
• molecules move slower decrease collisions between enzyme & substrate

Concentration and Type of Substrate

Enzymes have a saturation point, i.e., once all the enzymes added are occupied by the substrate molecules, its activity will be ceased.  When the reaction begins, the velocity of enzyme action keeps on increasing on further addition of substrate. However, at a saturation point where substrate molecules are more in number than the free enzyme, the velocity remains the same.

The type of substrate is another factor that affects the enzyme action. The chemicals that bind to the active site of the enzyme can inhibit the activity of the enzyme and such substrate is called an inhibitor. Competitive inhibitors are chemicals that compete with the specific substrate of the enzyme for the active site. They structurally resemble the specific substrate of the enzyme and bind to the enzyme and inhibit the enzymatic activity. This concept is used for treating bacterial infectious diseases.

Salt concentration

Changes in salinity: Adds or removes cations (+) & anions (–)

• Disrupts bonds, disrupts the 3D shape
• Disrupts attractions between charged amino acids
• Affect 2° & 3° structure
• Denatures protein
• Enzymes intolerant of extreme salinity
• The Dead Sea is called dead for a reason