Enzymes PDF

Enzymes ENZYMES Definition Enzymes are biocatalysts mainly proteins in nature that regulate the rate of all biochemical reactions. Common Features - All are produced by living cells and can act outside these cells. - They are needed in very small amounts. - They...

Enzymes ENZYMES Definition Enzymes are biocatalysts mainly proteins in nature that regulate the rate of all biochemical reactions. Common Features - All are produced by living cells and can act outside these cells. - They are needed in very small amounts. - They accelerate the reaction without affecting its equilibrium (decrease energy of activation). - They are not changed chemically by the end of the reaction. - They are highly specific (act on a specific substrate or inter-related substrates). Nomenclature - Most commonly used enzyme names have the suffix “-ase” attached to the substrate of the reaction (for example, glucosidase, urease, sucrase), or to a description of the action performed (for example, lactate dehydrogenase and adenylyl cyclase). - Some enzymes retain their original trivial names, which give no hint of the associated enzymatic reaction, for example, trypsin and pepsin. Enzyme Specificity - Enzymes are highly specific in their action, interacting with one or a few specific substrates and catalyzing only one type of chemical reactions. - The specificity of enzymes is due to the nature and arrangement of the chemical groups at the catalytic site (see latter). This allows the enzyme to unite and activate only one substrate or a small number of structurally related substrates. Importance of enzyme specificity: - The relatively low specificity of the digestive enzymes allows only a few enzymes to digest all foodstuffs. The high specificity of intracellular enzymes allows metabolic pathways to work and to be regulated more properly. Chemical Nature - Except for ribozymes, virtually all enzymes are protein in nature; some are simple proteins while others are conjugated proteins (holoenzyme). - Holoenzyme refers to the active enzyme with its nonprotein component (cofactor), whereas the enzyme without its cofactor is termed an apoenzyme and is inactive. - Cofactors are organic (Coenzymes) or inorganic molecules (Metal ions) that are required for the activity of a certain conjugated enzymes. They participate in substrate binding or catalysis. Mechanism of Enzyme Action (induced fit model) The Active Site: - Enzyme molecules contain a special pocket or cleft called the active site. - The active site contains amino acid side chains that create a three-dimensional surface complementary to the substrate.  Enzymes - The active site binds the substrate, forming an enzyme–substrate (ES) complex; ES complex is converted to an enzyme–product (EP) complex that subsequently dissociates to enzyme and product. - The induced fit model states that when substrates bind to an enzyme, they induce a conformational change analogous to placing a hand (substrate) into a glove (enzyme). T1 Role of Enzymes in Catalysis A- Lower activation energy Activation energy of Uncatalyzed reaction  Energy of activation, is the energy difference between that of the reactants and a T2 high-energy intermediate (transition state), that occurs during the formation of the product. Activation energy of enzyme catalyzed reaction  To allow a reaction to proceed rapidly , the enzyme provides an alternate A reaction pathway with a lower free energy of activation than that of G the un-catalyzed reaction. B B- Precise arrangement of chemical groups at the active site  The amino acids at the active site are arranged in a very precise manner so that only specific substrate or inter-related substrates can bind at the active site.  Also, the shape and the chemical environment inside the active site permit a chemical reaction to proceed more easily.  Usually serine, histidine, cysteine, aspartate and glutamate residues make up active site. Factors Affecting the Rate of Enzyme Catalyzed Reaction 1- Substrate Concentration [S] 2- Enzyme Concentration (E) 3- Concentration of inhibitors 4- Concentration of Cofactors [C] 5- Temperature 6- PH In studying the effect of any of the factors on enzyme catalysis, only one factor has to be varied at a time, all other factors being kept constant. The rate of the reaction should always be measured at the very beginning of the reaction, the so-called initial velocity (V0 or Vi ).  Enzymes 1- Effect of Substrate Concentration [S]  If all other conditions are kept constant, the velocity of the reaction increases as the substrate concentration [S] increases up to point where the enzyme is said to be saturated, the measured initial velocity or Vi increases to a maximum value Vmax.  The substrate concentration that produces half the maximal velocity is termed Michaelis constant or Km.  Smaller Km reflects higher affinity of the enzyme for its substrate and vice versa. Michaelis-Menten Equation The Michaelis-Menten equation describes the behavior of many enzymes as substrate concentration is changed. Vmax [S] Vi = Km + [S] Lineweaver-Burk Plot or Double-Reciprocal Plot The curve that describes the effect of substrate concentration is hyperbolic and it is quite difficult to estimate the Vmax as the value is never reached with any finite substrate concentration. This in turn makes it difficult to determine the Km value. It is easier to work with a straight line than a curve. One can transform the equation of hyperbola into an equation of a straight line by taking the reciprocal of both sides to yield the following: 1 Km 1 1 = Vmax + Vi [S] Vmax Lineweaver-Burk or double-reciprocal plot 1/ Vi Vmax Slope = Km / Vmax 1/2 Vmax Intercept on x-axis Intercept on y-axis = -1/ Km = 1/ Vmax Km1 Km2 [S] 0 1/ [S] 5- Effect of Temperature  At 0 °C enzyme action virtually stops due to the inhibition of movement and collision between the substrate and enzyme molecules.  As the temperature rises the velocity of the reaction increases due to increased kinetic energy of the molecules and increased collision between substrate and enzyme molecules, increasing the formation of ES complex.  Enzymes  The increase in the velocity of the reaction continues up to a point, “the optimum temperature”, beyond which any further increase in temperature causes a decrease in the reaction rate.  For most enzymes, the activity virtually stops at about 70 °C, due to denaturation of the enzyme protein, disrupting the organization of the catalytic site.  The optimum temperature for most animal enzymes is about 37°C, while that of most plant enzymes is about 50 °C. 6- Effect of pH  Each enzyme has an optimum pH at which it shows maximal activity. Activity decreases as we go away from the optimum pH, it virtually stops about 2 units of pH above or below this pH.  Slight changes in pH causes marked changes in enzyme activity due to alteration of the charges on the substrate and on the catalytic site of the enzyme.  Extreme changes of pH cause denaturation and irreversible inhibition of enzyme action.  Most enzymes have an optimum pH between 5 and 9. There are some exceptions, for example, pepsin, a digestive enzyme in the stomach, is maximally active at pH 2, whereas other enzymes, designed to work at neutral pH, are denatured by such an acidic environment Inhibition of Enzyme Activity Any substance that can diminish the velocity of an enzyme-catalyzed reaction is called an inhibitor. Enzyme inhibition can be either reversible or irreversible. I- Reversible Enzyme Inhibition Most common types: A. Competitive inhibition B. Allosteric inhibition. A- Competitive Inhibitors (Substrate analogue inhibitors)  A competitive inhibitor is structurally similar to that of substrate. Hence, it competes with substrate to bind reversibly at active or catalytic site.  No Effect on Vmax: The effect of a competitive inhibitor is reversed by increasing [S]. At a sufficiently high substrate concentration, the reaction velocity reaches the Vmax observed in the absence of inhibitor.  Increase of Km: A competitive inhibitor increases the apparent Km for a given substrate. This means that, in the presence of a competitive inhibitor, more substrate is needed to achieve ½Vmax. Competitive inhibitor 1/ Vi Competitive inhibitor No inhibitor No inhibitor Vmax 1/2 Vmax Intercept on x-axis Intercept on y-axis = -1/ Km = 1/ Vmax Km1 Km2 [S] 0 1/ [S] Lineweaver-Burk or double-reciprocal plot  Enzymes Examples for competitive inhibitors 1-Allopurinol is a drug used in the treatment of gout. i. Gout is due to excessive production of uric acid. ii. Xanthine oxidase is an enzyme involved in the formation of uric acid from hypoxanthine. iii. Allopurinol is a structural analog of hypoxanthine. iv. Allopurinol blocks the formation of uric acid by inhibiting the enzyme xanthine oxidase. Hypoxanthin Allopurinol e 2-Sulfonamides are used in the treatment of bacterial infections. i. Bacteria synthesize folic acid from p-aminobenzoic acid (PABA). ii. Since these sulfonamide drugs contain sulfanilamide a structural analog of PABA, when used as chemotherapeutic agent, it blocks the synthesis of folic acid in bacteria which is essential for bacterial multiplication (sulfonamides act as a bacteriostatic agent). iii. Sulfonamides act as a competitive inhibitor for the enzyme involved in the formation of folic acid using PABA as substrate. Precursor Folic acid Bacterial growth Sulfonamide Block H2N COOH H2N SO2N PABA Sulfanilamide 3- Dicumarol and warfarin are used as anticoagulants because they are structurally similar to vitamin K (required for activation of blood clotting factors). 4- Statins are competitive inhibitors of the key enzyme of cholesterol synthesis (HMG- CoA reductase) thereby lowering plasma cholesterol levels. B- Allosteric Inhibitors  They are usually small organic molecules that bind to a specific site away from the catalytic site and produce conformational changes in protein structure that lead to decrease activity of the enzyme e.g. ATP for phosphofructokinase-1.  Allosteric inhibitors decrease the affinity of the enzyme to its substrate (increase Km value), decrease the maximal catalytic activity (decrease Vmax) or both. Feedback Inhibition: It is the inhibition of the activity of an enzyme in a pathway by the end products of this pathway. It may occur through binding of the end product with an allosteric site present on the regulatory key enzyme. This usually occurs at the earliest irreversible step unique to that particular pathway. This prevents accumulation of unwanted amounts of the  Enzymes metabolic end products. II- Irreversible Enzyme Inhibition: A- Inhibitors that exert their effects on enzymes 1. Inhibitors that denature proteins (These include strong acids, alkalis, alcohols and salts of heavy metals). 2. Antienzymes: Antienzyme is specific for the enzyme that binds with it and produces its inactivation. Example: Antithrombin (activated by heparin) inhibits blood clotting. 3. Inhibitors that block chemical groups (enzyme poisons) a) Inhibitors of the sulfhydryl group (SH group) i. Oxidizing agents. Example: Hydrogen peroxide (H2O2) ii. Salts of heavy metals. Example: Hg2+ combine with the negatively charged sulfur of the SH group (Enz-S- Hg -S-Enz). b) Inhibitors that block hydroxyl groups (OH group) Aspirin produces acetylation of the hydroxyl group of serine at the active site of cyclooxygenase enzyme (responsible for prostaglandin synthesis), explaining the anti-inflammatory and antipyretic actions of aspirin. B- Inhibitors that exert their effects on cofactors or prosthetic groups 1. Fluoride blocks the action of enzymes, which require Ca2+ & Mg2+ ions by chelating these ions in the form of salts e.g. chelation of Mg2+ in case of enolase (an enzyme of glucose oxidation), results in inhibition of the enzyme. 2. Cyanide and carbon monoxide inhibit the activity of cytochrome oxidase an enzyme of respiratory chain by blocking the iron of heme. Regulation of Enzyme Activity It is important to control the rate of different metabolic reactions to maintain cellular structures and functions under different conditions. Regulation of enzyme activity is achieved through different mechanisms. I. Changing the E absolute amount & II. Changing the catalytic activity of the E. I- Changing the Absolute Amount of the Enzyme Present The amount of the enzyme present is determined by its rate of synthesis and rate of degradation. 1- Control of Enzyme Synthesis This is mainly performed through inducers and repressors. Inducers are substances, which stimulate gene expression into proteins. In case of enzymes, the inducers may be the substrate of the enzyme or hormones. Repressors are substances, which inhibit gene expression into proteins. In case of enzymes, the repressor may be a metabolic product of the enzyme or hormones. 2- Control of Enzyme Degradation This is performed by controlling the rate of synthesis or activity of the enzyme responsible for their degradation  Enzymes II. Changing the Catalytic Activity of the Enzyme. 1- Activation of Zymogens (Proenzymes)  Many enzymes are formed in the form of proenzymes or zymogens. In this form they are inactive. Activation requires proteolysis (removal of a part of the polypeptide chain which masks the active site or substrate site).  Many of these enzymes after activation can activate its zymogen in a process termed autocatalytic activation (autocatalysis). 2- Allosteric Modifiers (Inhibitors and Activators)  Allosteric inhibition is explained before.  The binding of an allosteric activator with the allosteric site produces conformational changes in the protein structure of the enzyme, which result in increased velocity of the reaction.  Many cellular metabolic reactions are controlled in this way e.g. AMP acts as an allosteric activator for the phosphofructokinase-1 (PFK-1).  Allosteric activator increases the affinity of the enzyme to its substrate (decrease Km value), increases the maximal catalytic activity (increase Vmax) or both. 3- Covalent Modification  Phosphorylation and dephosphorylation: Many enzymes are activated by phosphorylation and inactivated by dephosphorylation and vice versa.  This means that the enzyme is present in two interconvertible forms (phosphorylated and dephosphorylated). The phosphate groups are usually attached to the hydroxyl group of amino acid residues (mainly serine or tyrosine) present in the polypeptide chain of the enzyme. Isoenzymes (Isozymes) These are a group of enzymes which are characterized by the following: 1. They catalyze the same reaction. 2. They have different polypeptide chains which are produced by different genes. 3. They are separated by electrophoresis (have different migration rate). 4. They have different affinity to the substrate. 5. They are usually affected in different ways by the different activators and inhibitors. 6. They are present in the same (different compartments) or different cells. Examples for isozymes include the following: I- Lactate dehydrogenase (LDH) It is tetrameric enzyme. It is formed of four protomers (subunits) of two types H and M. The tetrameric molecule is the only active form of the enzyme. The different subunits are combined to form five isozymes as follows: Isozymes of LDH Type of subunits  Enzymes 1- I1 (H4 or HHHH) 2- I2 (H3M1 or HHHM) 3- I3 (H2M2 or HHMM) 4- I4 (H1M3 or HMMM) 5- I5 (M4 or MMMM) H and M subunits are synthesized by distinct genes and are differentially expressed in different tissues. Estimation of LDH isozymes in plasma is of clinical importance. Isozyme 1 is mainly of cardiac origin and its level in plasma increases in cases of myocardial infarction. Isozyme 5 increases in plasma in cases of liver diseases (hepatic origin) or muscle diseases (muscular origin). II- Creatine Kinase (CK) or Creatine Phosphokinase (CPK) It is a dimer formed of two subunits termed M or B. It has three isozymes as follows: 1- CK1 (or CK-BB) present in brain tissues and its plasma level increases in case of brain infarction. 2- CK2 (or CK-MB) present in cardiac muscles more than in skeletal muscles, so its plasma level increases markedly in myocardial infarction. 3- CK3 (or CK-MM) present in skeletal muscles more than in cardiac muscles, so its plasma level increases markedly in muscle diseases. Clinically Important Enzymes and Diagnostic Applications Principle Sources of Enzyme Principal Source Clinical application Acid phosphatase Red cells and prostate cancer prostate Alanine Liver Hepatic parenchymal diseases aminotransferase Alkaline Liver, bone, intestinal phosphatase Bone diseases, hepatobiliary diseases mucosa and placenta Salivary glands and Parotitis Amylase pancreas Pancreatitis Aspartate Liver, skeletal muscle and Hepatic parenchymal disease, muscle aminotransferase heart and cardiac diseases Creatine kinase Muscle diseases and myocardial Skeletal muscle, and heart infarction Heart, liver, skeletal Hemolysis, Lactate muscle, erythrocytes, hepatic parenchymal diseases, tumor dehydrogenase platelets, lymph nodes marker