Unit 1: Enzymes

(Enzymes) (Unit-1) (Part-2) The term 'enzyme' was coined by F. W. Kühne, to designate as biological catalysts, which were previously called 'ferments.' The previous name of enzyme or ferment comes from the word 'fermentation.' Definition: "Enzymes are macromolecular biocatalysts which catalyze thermodynamically feasible biochemical reactions." Enzymes are simple or combined proteins acting as specific catalysts. Enzymes affect the life of an organism to such an extent that life can be universally called as manifestation of enzymes. The enzymes carry out more than 5,000 biochemical reactions inside the living cells at normal temperature and pressure and at specific pH (4.0-9.0). Enzymes generally speed reaction rates by factors between 10^8 to 10^22 times compared with man-made catalysts. Structure of Enzyme: Thousands of reactions are controlled by enzymes in the living cells without the formation of toxic by-products. The enzymes are large protein molecules generally having tertiary or quaternary structure having specific catalytic sites known as 'active sites.' Enzyme are always the fundamental units of the cell metabolism. Generally, the enzymes are proteins. Both most of the enzymes are active when they are attached to some non-protein components. In such case, the protein part of the enzyme is known as 'apoenzyme' and the non-protein part is called 'cofactor.' The apoenzyme + cofactor complex, the conjugated protein is known as 'holoenzyme' (i.e. cytochrome C, catalase, peroxidase, etc.). Apoenzyme + cofactor = holoenzyme However, the term holoenzyme can also be applied to enzymes that contain multiple protein units such as the DNA polymerase. Cofactors: A cofactor may again be of three types i.e.: i. Co-enzyme ii. Prosthetic enzyme iii. Metal activator i. Co-enzyme: When the non-protein part of the holoenzyme is readily removed by dialysis, then it is known as co-enzyme or co-substrate. It is a thermostable, dialyzable organic compound. They may be loosely attached to the apoenzyme or may be present in the cytoplasm. In comparison to the entire enzyme molecule, co-enzymes account for only one percent. Generally, they belong to the dehydrogenase group of enzymes. Example: Nicotinamide adenine dinucleotide (NAD) known as coenzyme-1, nicotinamide adenine dinucleotide phosphate (NADP) called coenzyme-2, some vitamins (B, C, K), ATP, etc. ii. Prosthetic groups: When the non-protein part is firmly attached to the apoenzyme, then it is called a prosthetic group. Some non-protein ions may be tightly bound in some enzymes and hence act as prosthetic groups. Example: Cytochrome oxidase, catalase, peroxidase, etc. The tightly attached flavin adenine dinucleotide (FAD) in the enzyme succinic dehydrogenase is also a prosthetic group. iii. Metal activators: When some metal ions bind to the non-protein part of an enzyme, then it is known as termed as metal activator. 99% of enzymes contain metal ions as integral parts of an enzyme. Hence it is known as metalloenzyme. For example: (a) Copper-containing enzymes: Tyrosinase, ascorbic acid oxidase, (b) Iron and magnesium containing enzymes: Nitrogenase. Some metal activators may be less tightly bound to the enzyme and are required as added metals for activity. e.g. Mg2+, Mn2+, Ca2+ and K+ etc. Features of Active Sites: The enzymes have specific catalytic sites known as active sites or active centres or catalytic sites or substrate sites. The tertiary and quaternary structure of the enzyme proteins are produced due to folding of the polypeptide chain or chains. Interaction of groups to make active site (S) having correct placement of groups and appropriate topology to accommodate and bind with specific substrate (S). The active site may be present at the surface of the enzyme protein e.g. Chymotrypsin or B on (beta) (End of the apoenzyme or RNA etc.) The active site is produced by the side chain of the amino acid (R group) or R-(rest of the molecule) group and arrangement of groups of flexibility of tertiary or quaternary structure to hold and orient the substrate molecules. In some enzymes additional sites are present called regulatory sites. At this site regulatory molecules may attach to activate or deactivate the enzyme, e.g. Rubisco enzyme having 8 regulatory subunits. Substrate specificity: The enzymes are specific in nature and action. They only catalyze definite or specific structure or group of related substrate. The specificity may be of four types: Absolute specificity Group specificity Optical specificity or Geometrical specificity 1. Absolute specificity: In this pattern, the enzyme acts only on one specific type of substrate molecule, i.e. carbonic anhydrase acts on H2O and CO2 to produce carbonic acid. ​ (carbonic acid) 2. Group specificity: In this pattern, some enzymes catalyze a structurally related group of compounds (common groups) i.e. series of hexoses like glucose, mannose, fructose, etc. are taken by the hexokinase enzyme where the substrates are phosphorylated by ATP. Glucose ATP Hexokinase Glucose-6-phosphate ADP Glucose+ATP Hexokinase ​ Glucose-6-phosphate+ADP 3. Optical or stereo-specificity: In stereo-specificity, enzymes may have optical specificity for D- or L-optical isomers. For example, L-amino acid oxidase acts on L-amino acid whereas D-amino acid oxidase only reacts with D-amino acid isomers. L-amino acid Oxidase α-keto acid​ Oxidase ​ α-keto acid+NH Oxidase α-keto acid ​ Oxidase ​ α-keto acid+NH 4. Geometrical specificity: Still other enzymes have specificities toward geometric (cis-trans) isomers. Example: Fumarase readily adds water across the double bond system of trans isomer, fumaric acid but is inactive toward cis isomer, maleic acid. Fumarase L-malic acid (cis form) Fumarase+H ​ O→L-malic acid (cis form) Mechanism of Enzyme Action Enzymes neither initiate the reactions nor affect the equilibrium ratio of reactants and products. Rather, enzymes accelerate the rate of reactions (10^8 to 10^12 times) in both directions to attain the equilibrium position. It has been realized that an enzyme must temporarily form a chemical bond or enter into a transient complex with the substrate whose reaction it catalyzes. The specificity of substrate recognition in both formation of the complex and transformation of the substrate is due to the presence of a special enzyme active site on the surface of the enzyme, mainly of protein. A group of active site amino acids constitute the active site and their side chains form weak chemical bonds with the substrate. A number of theories have been put forward from time to time. All this history leads to the idea that the combination or complex produces some kind of activation of the substrate molecule due to: Polarization Displacement Strain or other disturbance of the bonds involved in the reaction. All the theories put forward up till now may be grouped into three: Theory concerning activation by enzymes. The theory of enzyme hydrolysis. Modern theories. Out of the above theories, modern emphasis is on a model given by modern theorists. On these, are based the following hypotheses which are being discussed regarding the mechanism of enzyme action. Lowering the activation energy of substrates by enzymes: When substrates react to form products, an energy barrier must be overcome. This energy barrier is called energy of activation. This energy barrier is called activation energy. It can be defined as the extra amount of energy which free average molecules must absorb from outside so that they can acquire the activated state to react with the reactants. Therefore, the enzyme carries out biochemical reactions at normal temperature, pressure, and pH by decreasing the energy of activation of substrates. Mechanism of Enzyme Action (contd.) It is also noted that the energy of activation is lowered for the forward as well as for the backward reaction. Example: In a living cell, due to the presence of enzyme (ATPase), 10^8 million molecules of ATP are converted to ADP and back to ATP every second because the reaction is catalyzed by an enzyme which lowers the energy of activation. [Graph showing the energy of activation with and without enzyme] Lowering the activation energy of substrates by enzymes (contd.): [Graph and illustration indicating energy levels] Enzyme-substrate complex formation: Lock and Key Hypothesis: Enzymes temporarily form weak chemical bonds with the substrates. A group of 3-dimensional amino acids readily constitutes the active site and their side chains form weak chemical bonds with the substrate. E + S ⇌ ES ⇌ EP ⇌ E + P where: E = Enzyme S = Substrate ES = Enzyme-Substrate Complex P = Product EP = Enzyme-Product Complex A German chemist, Hermann Emil Fischer, proposed that the conformation of active sites is such that it is exactly complementary to the substrate and so a catalyst acts just as a key fits into only its own lock and works. This hypothesis is popularly known as the lock and key hypothesis. This theory is supported from the study of competitive inhibition, in enzyme inhibition, the inhibitor closely resembles the structure of the substrate molecule and both of them compete for the same active site on the enzyme molecule. If the active site is rigid and specific for a given substrate, reversible or competitive inhibition cannot be considered, as the inhibitor is different from the substrate and could not fit well. Thus, this model fails to explain the reversible binding and stabilization of the transition state induced by the enzyme activity. Induced fit hypothesis: As contrasted to a rigidly arranged active site, Daniel Koshland (1958) found evidence that the active site of the enzyme can be induced by close approach of the substrate (or product) to undergo a change in conformation (shape) that allows a better combination between the two (enzyme and substrate or product). This idea is now widely known as induced fit theory hypothesis. Apparently, the active site of the substrate also changes its shape during many cases of induced fit, thus allowing a better interaction of the complex. The shape of the enzyme-substrate complex may be flexible and adaptable, and the substrate undergoes local changes. In some cases, the substrate may also change its shape slightly as it enters the active site. The active site continues to change until the substrate is completely bound, at which point the final shape and charge distribution is determined. [Diagram illustrating the induced fit model] Michaelis-Menten Equation: In biochemistry, Michaelis-Menten kinetics is one of the best-known models of enzyme kinetics. It is named after German biochemist Leonor Michaelis and Canadian physician Maud Menten. The model takes the form of an equation describing the rate of enzymatic reactions, by relating reaction rate v to the concentration of a substrate [S]. Its formula is given below: Here, Vmax = maximum rate achieved by the system, which happens at saturating substrate concentration. Km = Michaelis constant (its value is numerically equal to substrate concentration at which the reaction rate is half of Vmax). Biochemical reactions involving a single substrate are often assumed to follow Michaelis-Menten kinetics, without regard to the model's underlying assumptions. Enzyme Inhibition: Enzyme inhibition means the decrease in the rate of any enzyme-catalyzed reaction, which is done by inhibitors. Inhibitors try to prevent the normal functioning of enzymes and substrates, thereby affecting the process of "enzyme catalysis." The inhibitors are generally specific chemicals. Enzyme inhibition may be broadly two types: a) Irreversible Inhibition: Such type of inhibition may occur by irreversible inhibitors which combine or destroy a functional group on the enzyme molecule that is needed for enzyme activity, e.g., isopropyl fluorophosphate (DFP) which inhibits choline esterase enzyme. b) Reversible Inhibition: This type of enzyme inhibition occurs by reversible inhibitors which do not form any permanent damage on the enzyme molecule. The effects can be removed from the medium, enzyme activity becomes normal, and its functioning proceeds. We can use freely soluble or synthetic compounds as products of the reaction for inhibition. Two types of reversible inhibition are there, i.e., competitive and non-competitive inhibition. Competitive Inhibition: These types of inhibition are done by competitive inhibitors which have close resemblance to the substrate molecule. These are reversible inhibitors that get bound to the active site of the enzyme instead of a specific substrate, thus forming an enzyme-inhibitor (EI) complex rather than enzyme-substrate (ES) complex. Thus, this type of inhibitor competes with the substrate for the active site of the enzyme. The formation of the EI complex depends on the concentration of the inhibitor. Many bacteriostatic drugs act as competitive inhibitors, e.g., sulfa drugs that strongly inhibit the growth of bacteria containing the enzyme para-aminobenzoic acid. Non-Competitive Inhibition: Such inhibition is done by non-competitive inhibitors which are not nearly substrate-specific. These inhibitors bind at a site other than the catalytic site of the enzyme molecule. Thus, there is no competition for the substrate molecule. They alter the configuration of the enzyme so that the rate of enzyme catalysis is decreased. In this case, the substrate molecules also bind with the normal active site of the enzyme. The inhibition of enzyme E is not reversed even by increasing substrate concentration. Example: Many heavy metals like Ag²⁺, Hg²⁺, Pb²⁺ act as non-competitive inhibitors. B. Competitive Inhibitors: Chelating agents also act as non-competitive inhibitors. Uncompetitive Inhibition: This form of inhibition occurs when the inhibitor binds reversibly only with the ES complex to form an ESI (Enzyme-Substrate-Inhibitor) complex. ES unable to convert to any product. These types of inhibitors are most effective at high substrate concentration. Ex: When enzyme catalysis. This type of inhibition cannot be overcome by increasing substrate concentration but can be overcome by removing inhibitors or some other physical or chemical means. Factors Affecting Enzyme Activity: The rate of enzyme-catalyzed reactions is affected by the following factors: Substrate concentration Temperature pH Inhibitors Redox potential Water ions 1. Substrate Concentration: The rate of enzymatic reaction is enhanced with the increase in substrate concentration. However, an increase alone does not affect greatly unless further increased in substrate concentration. This point is called saturation when all enzyme molecules are active. Enzyme is saturated fully with the substrate. Ex: In some rare cases, enzyme activity may be decreased at high substrate concentration. This is called substrate inhibition.