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Enzymes: The Biological Catalysts Definition: Enzymes are biologic polymers that catalyze biochemical reactions. The vast majority of enzymes are proteins; however there are some catalytic RNA molecules called ribozymes. Enzymes are effective and highly specific catalysts: 1. Enzymes catalyze the conversion of one or more compounds called substrates into one or more compounds called products. 2. Enzymes accelerate (speed up) the rate of reaction by a factor of at least 106 . 3. Like all catalysts, enzymes are neither, consumed or altered after catalysis. 4. Enzymes are highly selective catalysts; they are specific for the type of reaction catalyzed and for a single substrate or a set of closely related substrates. RNA as an Enzyme Although enzymes are considered to be proteins, enzyme activity has recently been found in ribonucleic acid (RNA) in certain organisms. Enzyme Catalysis: The enzyme (E) has a reactive site (called active centre) which binds the reactant (or substrate (S)) by non-covalent interactions. The reaction starts by substrate binding to the enzyme to form ES complex, then reaction proceeds with the formation of the product (P): 1 Why are Enzymes Specific? Enzymes are very specific, and it was suggested by Emil Fischer in 1894 that this was because both the enzyme and the substrate possess specific complementary geometric shapes that fit exactly into one another. This is often referred to as "the lock and key" model. Lock & Key Model: The Lock and Key theory of enzyme activity states that every enzyme has a specific shape which allows its active site to fit with a specific substrate. Induced Fit Model The lock and key model has been modified by Daniel Koshland in 1958 by the induced fit model, which states that an enzyme can change its shape slightly to accept a fit with a substrate. 2 Mechanism of Enzyme Catalysis: Enzymes increase the rate of reaction by decreasing the energy of activation of the reactant 3 Classification of Enzymes: Enzymes are divided into six (6) major classes: 1. Oxidoreductases: involved in oxidation and reduction reactions, e.g. oxidases, dehydrogenases, oxygenases, peroxidases. 2. Transferases: transfer functional groups, e.g. amino or phosphate groups, e.g. aminotransferases, 3. Hydrolases: catalyze hydrolysis of the substrate, e.g. lipase, maltase, protease. 4. Lyases: add or remove elements of water, ammonia, or CO2 to form double bonds, e.g. decarboxylases. 5. Isomerases: catalyze the rearrangements of atoms within a molecule to give its isomer, e.g. glucose to fructose 6. Ligases: join 2 molecules, e.g. carboxylases and synthetases. Enzyme Cofactors: The majority of enzymes require the presence of some components to help them catalyze the reaction, these components are called cofactors. Cofactors can be classified into 3 groups as follows: 1. Coenzymes: These are organic compounds with low molecular weight, heat stable, loosely attached to the enzyme molecule, therefore can be separated easily by simple dialysis. Examples: NAD+, NADP+, B6-P. 2. Prosthetic groups: These are also low molecular weight organic compounds, which are firmly attached to the enzyme protein, therefore they are not separated by dialysis. Examples: FAD, heme. 3. Metal activators: These are inorganic monovalent and divalent cations such as K+, Mn2+, Ca2+, Zn2+, and Mg2+; these cations may be either loosely or firmly attached to the enzyme protein. 4 Holoenzyme, Apoenzyme and Coenzyme: Holoenzyme is the term used to describe the whole enzyme molecule which may be composed of an enzyme protein and a coenzyme or a prosthetic group. In this case, the enzyme protein is called apoenzyme. Therefore: Holoenzyme = Apoenzyme + Coenzyme (or Prosthetic group) Remember that: Neither Apoenzyme nor Coenzyme alone is catalytically active. Only when they are both combined together they become catalytically active. Proenzymes (Zymogens): Proenzymes (or zymogens) are inactive form of some enzymes produced by some cells, to become active in a second site like intestine and stomach. Examples: Pepsinogen (inactive form) is produced by gastric mucosal cells, and in the stomach, it is activated to pepsin by gastric acidity by active pepsin (Auto-activation): Pepsinogen Pepsin (Inactive) + HCl (Active) (or Pepsin) Isoenzymes: Isoenzymes mean a group of enzymes which catalyze one and the same reaction but they differ in physical, chemical, immunological and electrophoretic properties. Examples: (a) The enzyme lactate dehydrogenase (LDH) occurs in different forms, which can be separated from each other by electrophoresis into 5 types: LDH1, LDH2, LDH3, LDH4 & LDH5 which come from different organs; LDH1 is found in the heart and LDH5 occurs in the liver. Determination of the level of 5 either of these isoenzymes in blood is of value in the diagnosis of liver and heart diseases. (b) Creatine phosphokinases (CK) also have different isoenzyme forms. These isoenzymes are of value in the diagnosis of muscle, heart and brain diseases. Factors that Affect the Rate of Enzyme-Catalyzed Reactions 1. pH: A change in pH changes the rate of enzyme-catalyzed reaction. A bellshaped curve is obtained when the rate of the reaction is plotted against pH with an optimum pH at which the rate is optimum. Changes in pH can change the ionization of the substrate or the catalytic site of the enzyme.\ 2. Temperature: The rate of an enzyme-catalyzed reaction increases with increasing 6 temperature up to an optimum point (called optimum temperature), then it decreases because of denaturation of the enzyme protein 3. Substrate Concentration: The rate of enzyme-catalyzed reaction is increased with the increase of substrate concentration [S] till a point is reached (point of saturation), when there is no further increase in the reaction rate with the increase in substrate concentration. This observation can be explained as follows: a) At low substrate concentration [S]: the active sites (centers) of the enzyme are still not saturated with substrate molecules and addition of substrate increases [ES] complex gradually. b) At the point of saturation with the substrate: 7 As the substrate concentration increases, the sites become saturated and as substrate is added no further increase in the [ES] with no further increase in the rate of reaction and the result is the following graph: Vmax = Maximal velocity or Maximal rate of the reaction. KM = The substrate concentration that gives half maximal velocity (or 1/2 Vmax). KM is a good measure of enzyme affinity towards its substrate. This means that , the smaller the KM the higher the affinity and vice versa. 4. Concentration of the Product (P) of the reaction: Most enzyme-catalyzed reactions are reversible, therefore, if the products are not properly removed from the cell, they will inhibit the reaction rate. 5. Physical agents: Enzyme activity is seriously affected by some physical agents that cause denaturation of proteins, e.g. ultrasonic vibrations, x-ray, ultraviolet light (u.v), repetitive freezing and thawing, etc …. 6. Inhibitors: Many compounds can combine with enzymes although they are not 8 substrates, therefore, they may block the catalytic function of the enzyme. These compounds are called inhibitors. There are 2 main types of inhibitors: A. Competitive inhibitors B. Non-competitive inhibitors. A. Competitive Inhibitors: These are compounds which compete with the natural substrate for the biding with the active site of the enzyme, therefore decreasing the catalytic activity of the enzyme, e.g. the enzyme succinate dehydrogenase (SDH) oxidizes succinic acid to fumaric acid. Addition of malonic acid, which has a chemical structure similar to succinic acid, will cause inhibition of this enzyme. Such inhibition can be reversed by increasing the concentration of the substrate succinic acid. This shows that there is competition between the inhibitor (i.e. malonic acid) and the substrate (i.e. succinic acid) to bind the active site of the enzyme. This competition is due to similarity in chemical structure between succinc acid and malonic cid. Succinic Acid Malonic Acid 9 B. Non-competitive Inhibitors: These are compounds which combine irreversibly with the active center of the enzyme and is not displaced with increasing substrate concentration; Examples: Iodoacetate react with SH – groups essential for the catalytic activity of some enzymes: Enzyme – SH + ICH2- COOH Active enzyme Enzyme-S-CH2-COOH iodoacetic + HI Inactive enzyme Enzyme Inhibitors used as Drugs Inhibitors Target Enzyme Effect Allopurinol Xanthine oxidase Treatment of gout Aspirin Cyclooxygenase Anti-inflammatory 5-Flurouracil Thymidine synthetase Anticancer Lovastatin HMG-CoA reductase Cholesterol-lowering agent Penicillin Transpeptidase Antibacterial Capoten ACE inhibitor Antihypertensive agent Enzymes in Clinical Diagnosis Name of Enzyme Diagnostic Uses Acid phosphatase (AP) Prostate cancer Alanine aminotransferase (ALT) Liver damage, hepatitis Alkaline phosphatase (ALP) Liver disease, bone disease Amylase (AMS) Acute Pancreatitis Creatine kinase (CK) Muscle disorder, heart attack Lactate dehydrogenase (LDH) Heart attack (LD1), Liver disease (LD5) 10