Enzymes PDF

Enzymes Enzymes Enzymes are protein catalysts that increase the rate of chemical reaction in biological systems without changing themselves. Note: All enzymes are proteins except some types of RNAs which act as enzymes usually catalyzing the cleavage and synth...

Enzymes Enzymes Enzymes are protein catalysts that increase the rate of chemical reaction in biological systems without changing themselves. Note: All enzymes are proteins except some types of RNAs which act as enzymes usually catalyzing the cleavage and synthesis of phosphodiester bonds. These types of RNAs are called ribozymes. Substrate: It is the substance upon which the enzyme acts Cellular distribution of enzymes: 1- Intracellular enzymes: These enzymes are produced and act inside the cells e.g. metabolic enzymes (ALT, AST) 2- Extracellular enzymes: These enzymes are produced inside the cells but act outside the cells e.g. digestive enzymes (pepsin, α-amylase). - Enzymes are mainly synthesized in cytosol or ER (may stay, or transport to other organelles). - A relatively small numbers of enzymes are synthesized and act in the mitochondria. - Enzymes are present in all body cells. - Enzymes are found in both the extracellular fluids (plasma, CSF), and intracellular compartments. - Enzymes are distributed in cell membrane, cytosol, mitochondria, Lysosome, microsome, and nucleus….etc. Enzyme activity is expressed in: International unit (IU): It represents the amount of enzymes that catalyzes the conversion of one micromole (μmol) of substrate to product per minute. 1 enzyme U = 1 μmol min⁻¹ Katal: It represents the amount of enzymes that catalyzes the conversion of one mole of substrate to product per second. 1 Katal = 1 mol s⁻¹ Nature of Enzymes: Most enzymes are protein in nature. Enzymes can exist as simple enzyme or holoenzyme 1. Simple enzyme: It is made up of only protein molecule (pancreatic ribonuclease) 2. Holo enzyme is made up of protein part (apoenzyme) and non-protein part (cofactor). Cofactors may be organic compounds (vitamins) or inorganic metal ions (Fe²⁺, Zn²⁺, Mg²⁺,……) 1  Enzymes Organic molecules are divided into prosthetic groups and coenzymes - A prosthetic group describes a small organic molecule bound to the apoenzyme by covalent bonds. - A coenzyme describes a small organic molecule bound to the apoenzyme by non- covalent bonds. - A Coenzyme is a thermostable organic compound that is loosely attached to the enzyme. Coenzymes are derivatives of vitamins without which the enzyme cannot act. - Coenzyme can accept or donates a particular group during the reaction. 2  Enzymes Division of coenzymes: A. Codehydrogenase B. Group transferring coenzymes Vitamin B₃ (Niacin): 3  Enzymes Vitamin B₂ (Riboflavin): Glutathion: It is tripeptide (glutamate-cysteine-glycine). It acts as hydrogen donor e.g. insulinase enzyme (insulin-glutathion transdehydrogenase) inactivates insulin. Vitamin C (L-ascorbic acid): - It acts as reducing agent (hydrogen carrier). It acts as coenzyme for dopamine oxidase and polyphenol oxidase. Coenzyme Q (Ubiquinone): - It has a similar structure to vitamin K. It acts as hydrogen carrier in respiratory chain (electron transport chain), which is used in oxidative phosphorylation. 4  Enzymes Lipoic acid: - It acts as hydrogen carrier in oxidative decarboxylation. It acts as coenzyme for pyruvate dehydrogenase complex enzyme and a-ketoglutarate dehydrogenase complex enzyme. B. Group transferring coenzymes: Coenzyme A (CoASH): - It acts as a carrier of acyl group of fatty acids. It activates fatty acids to fatty acyl CoA. - It acts as coenzyme in oxidative decarboxylation. 5  Enzymes - Pyridoxal phosphate (Co-transaminase): - It acts as coenzyme in transamination, decarboxylation, Desulfuration of amino acids. - It acts as coenzyme for sGPT and sGOT. Vitamin B1 (Thiamin pyrophosphate TPP) = Cocarboxylase or Codecarboxylase - It acts as coenzyme in oxidative decarboxylation. Vitamin B7 (Biotin or Cocarboxylase): - It acts as CO₂ carrier in the fixation of CO₂ reactions. 6  Enzymes Coenzyme Active form Function Type of reaction Example of enzyme Folic acid Tetrahydrofolate (THF) Carrier of C atom Transport of Serine containing ( CH₃, CH₂, CHO, hydroxymethyl groups CH=NH) groups transferase Vitamin B12 Deoxyadenosylcob- CH₃ group carrier Transport of Methylmalonyl alamine methyl group CoA mutase Adenosinetriph Phosphate group Transport of Glucokinase -osphate (ATP) donor phosphate group UDP-glucose UDP-glucose glycogen Transport of Glycogen (active glucose) synthesis glucose synthase UDP-galactose UDP-galactose Lactose synthesis Transport of Lactose galactose synthase UDP-glucuronic UDP-glucuronic acid Muco-poly Transport of Glucuronyl acid saccharides glucuronic acid transferase synthesis Cytidinediph- CDP-choline Activation of Transport of Choline osphate (CDP) choline base choline in transferase lecithin synthesis Active S-Adenosyl Methyl group Transport of Methyl methionine methionine (SAM) donor methyl group transferase reactions Active sulfate Phosphadenosylph- Sulfate group Transport of Sulfate osphosulphate (PAPS) donor sulfate group transferase Metal ions in enzymes: - Nearly 1/3 of known enzymes require metal ions like Ca²⁺, K⁺, Mg²⁺, Fe²⁺, Cu²⁺, Zn²⁺, Mn²⁺ and Co²⁺ for their activity. Metal ions containing enzymes are divided into two types: 1-Metal-activated enzymes:- - A metal is loosely attached to apoenzyme only during the reaction. -The metal is easily dissociated from apoenzyme by dialysis process during purification (The metal is dialyzable) 7  Enzymes Example: Pyruvate carboxylase enzyme is activated by Mn²⁺. Metalloenzymes: - The metal is tightly attached to the apoenzyme and does not dissociated from the apoenzyme by dialysis process during purification. 1-The metal is essential for the formation of tertiary or quaternary structure of enzyme e.g. Ca²⁺-α-amylase, cytochrome, carboxypeptidase. 2- The metal ion may interacts with charged groups of the substrate forming a complex 3- The metal ion may forms a salt bridge between the apoenzyme and the coenzyme e.g. Zn²⁺ form a bridge between alcohol dehydrogenase enzyme and its coenzyme (NAD⁺). Examples: Metal Enzyme Calcium Lipase, lecithinase Magnesium Hexokinase, phosphofructokinase, enolase, glucose-6- phosphatase Iron Xanthine oxidase, cytochrome oxidase, peroxidase, catalase. Copper Cytochrome oxidase, lysyl oxidase, superoxide dismutase, tyrosinase. Manganese Hexokinase, enolase, phosphoglucomutase, glycosyl transferase Zinc Carbonic anhydrase, alcohol dehydrogenase, carboxy peptidase, alkaline phosphatase (ALP), lactate dehydrogenase (LDH). Molybdenum Xanthine oxidase 8  Enzymes A. Active site: Enzyme molecules contain a special pocket or cleft called the active site. This active site contains two sites: 1- Binding site at which the substrate is bound. 2- Catalytic site at which the reaction is catalyzed. The active site contains amino acid chains that create a three-dimensional surface complementary to the substrate. The active site binds to the substrate, forming an enzyme-substrate (ES) complex. ES is converted to enzyme-product (EP); which subsequently dissociates to enzyme and product. The active site of the enzyme may contain free hydroxyl group of serine, phenolic (hydroxyl) group of tyrosine, SH-thiol (Sulfhydryl) group of cysteine or imidazole group of histidine to interact with there is substrates. Catalytic efficiency: Most enzyme- catalyzed reactions are highly efficient proceeding from 10³ to 10⁸ times faster than uncatalyzed reactions. - Typically each enzyme molecule is capable of transforming 100 to 1000 substrate molecule in to product each second. - Enzyme turnover number refers to the number of molecules of substrate converted to products per time unit per enzyme molecule. (Carbonic anhydrase is one of the fast enzymes in human body). 9  Enzymes Nomenclature of enzymes: 1- Trivial name gives no idea of source, function or reaction catalyzed by the enzyme. Example: trypsin, thrombin, pepsin. 2- Recommended name: a- Most commonly used enzyme names have the suffix –ase – attached to the substrate of the reaction ex: glucosidase, urease, sucrase. b- Description of the action performed example: lactate dehydrogenase, succinate dehydrogenase and, pyruvate decarboxylase. 3- Enzyme Commission number: Each enzyme code consists of the letters "EC" followed by four numbers: The first number indicates the type of reaction. The second number indicates the function group. The third number indicates the coenzyme. The fourth number indicates the substrate e.g. alcohol dehydrogenases (EC 1.1.1.1). Specificity: Enzymes are specific for their substrates. Specificity of enzymes is divided into: 1-Absolute specificity (Substrate specificity):- It is also called high specificity, this means that one enzyme catalyzes or acts on only one substrate. For example: Urease catalyzes hydrolysis of urea but not thiourea. Enzyme Sucrase Maltase Lactase Arginase Uricase Carbonic anhydrase Substrate Sucrose Maltose Lactose Arginine Uric acid Carbonic acid 2-Group specificity (Moderate specificity):- It is also called structural specificity. - The enzyme will act only on molecules that have specific functional groups, such as peptide, amino, phosphate and methyl groups. 10  Enzymes - Example: pepsin enzyme hydrolyzes the peptide bond adjacent to aromatic amino acids (phenylalanine, tyrosine, and tryptophan). - Trypsin enzyme hydrolyzes the peptide bond adjacent to basic amino acids (Lysine, Arginine, Histidine) - Aminopeptidase hydrolyzes the peptide bond adjacent to the free amino group - Carboxypeptidase hydrolyzes the peptide bond adjacent to the free carboxyl group - Hexokinase catalyzes the phosphorylation of hexose sugars e.g. glucose, galactose and mannose. 3- Relative Specificity (Low specificity): It is also called bond specificity. These enzymes act on several substrate that have the same chemical bond. Examples: - Amylase enzyme acts on α (1,4) glycosidic bond in starch, dextrins and glycogen - Lipase enzyme acts on an ester bond in any type of TAG (Tri Acyl Glycerol). 4- Optical specificity (Stereo specificity):- some enzymes are specific to only one optical isomer and not the other. For example: L-amino acid oxidase enzyme acts only on L-amino acid but not D-amino acids and arginase catalyzes the hydrolysis of L-arginine but not D-arginine. Maltase catalyzes the hydrolysis of - but not -glycosides. 5- Dual specificity: Some enzymes act on 2 substrates in the same reaction. Example: - The enzyme may acts on one substrate by two different reactions e.g. isocitrate dehydrogenase catalyzes oxidation and decarboxylation reactions of citrate. 11  Enzymes Zymogens (Inactive form of enzyme): - Some enzymes are produced as inactive form which can be activated when they are required. Such types of enzymes are called zymogens (or proenzymes). - Zymogen is inactive because its active site is masked by a segment of a polypeptide chain. - Zymogens are activated by cleaving the polypeptide chain to open the active site. Activation of zymogens can occur by one of the following methods: 1) By HCl 2) By other enzyme 3) Auto activation Classification of Enzymes: Enzymes are classified on the basis of the reactions they catalyze into six major classes: Class I. Oxidoreducatases: - These enzymes catalyze oxidation reduction reactions. This group is subclassified into the following 4 subgroups: 1- Oxidase enzymes 2- Dehydrogenase enzymes 3- Peroxidase enzymes 4- Oxygenase enzymes A. Oxidases catalyze the removal of hydrogen from a substrate using oxygen as a hydrogen acceptor. They form water or hydrogen peroxide as a reaction product. 12  Enzymes 1- Some oxidases contain copper e.g cytochrome oxidase (hemoprotein). 2- Other oxidases are Flavoproteins e.g L-amino acid oxidase. B. Dehydrogenase cannot use oxygen as hydrogen carrier. 1-Many dehydrogenases depend on nicotinamide coenzymes (NAD⁺ and NADP⁺). 2- Other dehydrogenases depend on riboflavin (FAD). 3- Cytochromes may also be regarded as dehydrogenases (cyt b, cyt c, cyt a,a₃). C. Hydroperoxidases use H₂O₂ or organic peroxide as substrate. 1- Two type of enzymes found both in animals and plants fall into this category: peroxidases and catalase. 2- Peroxidases reduce peroxides using various electron acceptors such as ascorbate, quinones, reduced glutathione. 3- Catalase uses hydrogen peroxide as electron donor and electron acceptor. D. Oxygenases catalyze the direct transfer and incorporation of oxygen into a substrate molecule. 1- Dioxygenases incorporate both atoms of molecular oxygen into the substrate e.g. tryptophan pyrrolase (tryptophan oxidase), carotene dioxygenase (carotenase). 2- Mono-oxygenases (mixed-function oxidases, hydroxylases) incorporate only one atom of (O₂) into the substrate e.g. tyrosine hydroxylase (tyrosinase), phenyl alanine hydroxylase. 3- Cytochromes P450 are monooxygenases important for the detoxification of many drugs and for the hydroxylation of steroids e.g homogentisate dioxygenase and L-tryptophan dioxygenase. E. Superoxide dismutase protects aerobic organisms against oxygen toxicity. - Transfer of a single electron to O₂ generates the potentially damaging superoxide anion free radical (.O₂⁻). Example: Lactate-dehydrogenase 13  Enzymes L-ascorbate oxidase is an enzyme that catalyzes the chemical reaction. 2 L-ascorbate + O2 2 dehydroascorbate + 2 H2O Dehydrogenase Aerobic In abundance of oxygen forming H₂O₂ Examples: 1-L-aminoacid oxidase 2-D-aminoacid oxidase Anaerobic Examples: 1- Lactate dehydrogenase 2-Succinate dehydrogenase Oxidase Examples: 1- Ascorbic acid oxidase 2-cytochrome oxidase Reductase Examples: Glutathione reductase Peroxidase Examples: 1- Glutathione peroxidase 2- Catalase Oxygenase Mono- Example:1-Phenylalanine hydroxylase Oxidoreductase oxygenase 2-Tyrosine hydroxylase (tyrosinase) Dioxygenase Example:1- Tryptophan pyrrolase 2- Carotene dioxygenase (Carotenase) Class II. Transferases: These enzymes catalyze a transfer of a group other than hydrogen (methyl, acyl, amino or phosphate groups). - Enzymes catalyzing transfer of phosphate group are called Kinases. Examples: 14  Enzymes Class III. Hydrolases: These enzymes catalyze the hydrolysis of peptide, ester, glycosidic, phosphate bonds (cleavage of bond by addition of water). 1- Carbohydrases (maltase, lactase, sucrose and amylase) hydrolyze the glycosidic bond. 2- Lipases hydrolyze the ester bond of TAG. 3- Proteases (pepsin, trypsin) hydrolyze the peptide bond of proteins. 4- Phosphatases (glucose-6-phosphotase) hydrolyze the phosphoester compounds releasing phosphate group. Class IV. Lyases: These enzymes catalyze removal of groups from substances by mechanisms other than hydrolysis, leaving double bonds. Examples: 15  Enzymes Class V. Isomerases: These include all enzymes catalyzing interconversion of optical, geometric, or positional isomers. 1- Aldose-ketose isomerase 2- Epimerase 3- Mutase 4- Racemase 5- Cis-trans isomerase Class VI. Ligases or synthetases. These enzymes catalyze the linking of 2 compounds coupled to the breaking of a pyrophosphate bond in ATP or similar high energy trinucleotides: GTP, UTP … etc. These are enzymes catalyzing reactions forming C-O, C-S, C-N, and C-C bonds. Examples: 16  Enzymes Mechanism of enzyme action: The mechanism of enzyme action can viewed from two different perspectives: 1- Energy changes 2- chemistry of the active site 1- Energy changes occur during the reactions: - All chemical reactions have an energy barrier separating the reactants and the products; this barrier is called the free energy of activation. Activation energy: It is the amount of energy required to raise all the molecules in one mole of substrate to the transition state (T*). - The effect of enzyme is to decrease the energy of activation by providing alternate reaction pathway with a lower free energy of activation. Active site: The specificity of an enzyme is determined by: 1- The functional group of the substrate (or product). 2- The functional group of the enzyme and its cofactor. 3- The physical proximity of these various functional groups. a) During the enzyme action there is a temporary combination between the enzyme and its substrate forming enzyme-substrate complex, this occurs at active site of enzyme. b) This is followed by dissociation of this complex into enzyme again and products. Two theories have been proposed to explain the specificity of enzyme action: 1-Emil Fischer’s model Lock and Key theory 1890. The active site of the enzyme is complementary in conformation to its substrate, so that the enzyme and substrate recognize one another. 17  Enzymes 2- The induced fit theory: In 1958, Daniel Koshland, postulated another model. The enzyme changes its shape upon binding the substrate, so that the conformation of enzyme is complementary to its substrate only after the substrate is bound. Enzyme kinetics is the study of rates of chemical reactions that involve enzymes. Velocity of reaction: It is the increase of the concentration of products with time or it is the decrease of the concentration of substrate with time. Initial velocity: The velocity at which the reaction proceed, is measured as ↑↑[products] with time or ↓↓[substrate] with time. The graphic relationship of the [product] versus time is hyperbolic. - The decline in the rate of the reaction may be due to depletion of the substrate, inhibition of the 18  Enzymes enzyme by its product or denaturation of the enzyme. - Initial velocity (Vₒ) of the reaction: It is the initial portion of the reaction where the increase in the concentration of the product is correlated constantly with time. Factors Affecting Enzyme Activity: Physical and chemical factors are affecting the enzyme activity. These include: 1-Temperature 2- pH 3- Enzyme concentration 4- substrate concentration 5- concentration of coenzyme 6- Enzyme activator 7- Enzyme inhibitor 1-Temperature: Starting from low temperature as the temperature increases to certain degree (37°) the activity of the enzyme increases. The optimum temperature for the enzyme is the temperature at which an enzyme shows maximum activity. For most body enzymes the optimum temperature is around 37°C, which is body temperature. 2-Effect of pH: The concentration of H⁺ affects reaction velocity in several ways. First, the catalytic process usually requires that the enzyme and substrate have specific chemical groups in an ionized or unionized state in order to interact. For example, Catalytic activity may require that an amino-group of the enzyme be in the protonated form (-NH₃⁺). At alkaline pH this group is deprotonated (-NH₂) and the rate of reaction therefore declines. Extreme pH can also lead to denaturation of the enzyme, because the structure of the catalytically active protein molecule depends on the ionic character of the amino acid chains. 19  Enzymes Optimum pH for the enzyme is the pH at which an enzyme shows maximum activity. For example, pepsin, a digestive enzyme in the stomach, has maximum action at pH 2. Enzyme Lipase Pepsin Acid Arginase Salivary α- (pancreas) phosphotase Amylase Optimum pH 7.5-8.0 1.5-2 4.5-5 9.5-9.9 6.7-7 3- Enzyme Concentration: The rate of the reaction is directly proportional to enzyme concentration at all substrate concentration. For example, if the enzyme concentration is doubled, the initial rate of the reaction (Vₒ) is doubled. 4- Concentration of substrate: The initial velocity of a reaction is directly proportional to the amount of substrate present till a maximum point (maximum velocity Vmax) where any further increase in the amount of substrate concentration does not increase rate of reaction. a) At low [substrate], not all enzymes are saturated, so the rate of reaction will increase. b) At higher [substrate], all enzyme molecules get saturated with substrate and any more increase of [substrate] will result in no increase in the rate of the reaction. Michaels and Menten equation: 1- This equation describes the dependence of reaction velocity on substrate concentration Michaelis and Menten proposed that in any enzymatic reaction, the enzyme (E) combines with substrate (S) to form an enzyme-substrate complex (E-S). (ES) complex then dissociates to form product (P) and enzyme (E) back. 20  Enzymes Km is numerically equal to the substrate concentration of which the reaction velocity equal to ½ Vmax i) Km is characteristic of an enzyme and a particular substrate, and reflects the affinity of the enzyme for that substrate. ii) Km values vary from enzyme to enzyme and used to characterize different enzymes. iii) Km values of an enzyme helps to understand the nature and speed of the enzyme catalysis. iv) Small Km value (Low km) reflects a high affinity of the enzyme for substrate because a low concentration of substrate is needed to half saturate the enzyme- that is reach a velocity of ½ Vmax. v) High Km value (high Km) reflects a low affinity of enzyme for substrate because a high concentration of substrate is needed to half saturate the enzyme. 5-Concentration of coenzyme: In conjugated enzymes, increase the concentration of coenzyme leads to increase the rate of reaction. 6- Concentration of metal ion (activators): ↑↑[Ca²⁺] leads to ↑↑ the activity of thrombokinase enzyme ↑↑[Cl⁻] leads to ↑↑ the activity of salivaryα-amylase 21  Enzymes 7- Presence of inhibitors: Inhibitor is the substance that decreases the enzyme activity and in turn decreases the velocity of enzymatic reactions. Example: Malonic acid decrease the activity of succinate dehydrogenase enzyme which catalyzes the conversion of succinic acid to fumaric acid Enzyme Inhibition: Any substance that can diminish the velocity of an enzyme- catalyzed reaction is called an inhibitor and the process is known as inhibition. There are two major types of enzyme inhibition, Irreversible and Reversible: Reversible Inhibition: This type of inhibition can be Competitive, Non-competitive and uncompetitive Competitive Inhibition: In competitive inhibition the inhibitor and substrate compete for the same active site on the enzyme as a result of similarity in structure. - The enzyme substrate complex will be broken dawn to products (E+S→[ES]→E+P) whereas enzyme inhibitor complex; (EI) will not be broken down to products. Example 1: A classic example is Malonate that competes with succinate and inhibits the action of succinate dehydrogenase to produce fumarate in the Krebs cycle 22  Enzymes Eg.2 Allopurinol used for the treatment of Gout Allopurinol Inhibits Xanthine oxidase enzyme by competing with hypoxanthine. This competition blocks the conversion of hypoxanthine and xanthine, to uric acid and result in lower serum urate levels. 3- Sulfanilamide competes with p-amino benzoic acid for dihydrofolate synthetase enzyme which catalyzes the synthesis of folic acid in bacteria 4- Methotrexate competes with dihydrofolic acid for dihydrofolate reductase 5-Physostigamine competes with acetyl choline for choline esterase enzyme 6- Acetazolamide (inhibitor) competes with carbonic acid for carbonic anhydrase enzyme 7- Dicumarol (inhibitor) competes with vitamin K for prothrombin synthesis Effect of Competitive inhibitors: 1. Effect on Vmax: Vmax does not affect (Vmax is constant) 2. Effect on Km: Km ↑↑ 23  Enzymes Non-Competitive Inhibition: This type of inhibition occurs when the inhibitor and substrate bind to different sites on the enzyme. Non-competitive inhibitors can bind reversibly either to the free- enzyme or the ES complex to form the inactive complexes EI and ESI (Enzyme substrate Inhibition). -The reaction may be slowed but not stopped -There is no structural similarity between inhibitor and substrate. An Example: is the inhibition of L-threonine dehydratase by L-isoleucine. Such type of enzyme is called Allosteric Enzyme, which has a specific sites or allosteric site other than the substrate-binding site. 1. Effect on Vmax: (Vmax ↓↓) 2. Effect on Km: (Km is constant) Uncompetitive Inhibition Uncompetitive Inhibitor binds only to ES complex at locations other than the catalytic site. In this case apparent Vmax, and Km decreased. Irreversible Inhibition: The type of inhibition that cannot be reversed by increasing substrate concentration or removing the remaining free inhibitor is called Irreversible inhibition. Examples: 1- Heat, X-rays, ultraviolet rays, strong (acids and alkalies), severe agitation, repeated freezing and thawing, and heavy metals e.g Hg, Ag, Pb are non-specific irreversible inhibitors of all enzymes. 2- Organo-phosphorus compounds like Malathion and Parathioon pesticides-inhibits acetyl cholinesterase. 24  Enzymes 3- Inhibitors of SH group in enzymes e.g. Alkylating agent e.g iodoacetic acid reacts with thiol (SH) group of enzymes Enzyme-SH + I-CH₂-COOH → Enzyme-S-CH₂-COOH + HI 4- Toxic gases used in chemical war e.g. lewisite binds to thiol (SH) group of enzymes causing inhibition of these enzymes. 5- Oxidizing agents e.g. K₃[Fe(CN)₆] combines with thiol (SH) of enzyme. Anti-enzymes: These substances are secreted by the living cells and irreversibly inhibit the enzyme activity e.g. 1- Ascaris worm protects itself in GIT by secreting antipepsin and antitrypsin 2- Thrombin can be inhibited by enzyme antithrombin III in plasma 3-Mucin lining the stomach wall secrets antipepsin to protect stomach against pepsin 4- The production of the antibodies against any enzyme by injecting this enzyme into specific animal e.g. rabbit Irreversible Inhibitors that block activators and coenzymes: These inhibitors chelate with the activators, and in turn inhibit the action of enzyme e.g. 1-Fluoride ion (F⁻) inhibits many enzymes such as enolase and clotting enzymes. This is because fluoride ion (F⁻) can precipitate Ca²⁺ which is required for the activation of clotting enzymes. Fluoride ion (F⁻) also can precipitate Mg²⁺ which is required for the activity of enolase enzyme. 2- Cyanide ion (CN⁻), azide ion (N₃⁻) and carbon monoxide (CO) can bind to Fe ions which are required for the activity of cytochrome oxidase enzyme. 3- Isoniazide (INH) drug used for the treatment of tuberculosis (TB). This drug combines with pyridoxal phosphate coenzyme which is required for the activity of transaminases enzymes 4- EDTA (Ethylene Diamine Tetr Acetic acid) forms a chelate with Mg²⁺ and Ca²⁺ and in turn enolase and kinases enzymes are inhibited. Note: Recently inhibitors are classified according to the site of action e.g. catalytic site, binding site, allosteric site 25  Enzymes Regulation of enzyme activity occurs by: 1-Amount of enzyme 2- Allosteric regulation 3- Feedback inhibition 4- Feedback regulation 5- Covalent modification (phosphorylation/dephosphorylation 1- Amount of enzyme present in the cell: This is determined by the rate of enzyme synthesis and adaptation Rate of enzyme synthesis is regulated by both of inducers and repressors Inducer is the substance that increase (induction) the protein (enzyme) synthesis e.g. after carbohydrate meal the plasma glucose level increases this leads to increase secretion of insulin which induces the synthesis of all key enzymes of glycolysis (glucokinase, phosphofructokinase and pyruvate kinase) Repressor is the substance that decrease the protein synthesis e.g. glucagon hormone decreases the synthesis of the key enzymes of glycolysis 2-Allosteric regulation: Enzyme molecule may has a regulatory site other than the active site, this site is called allosteric site and these enzymes known as allosteric enzymes Effectors are the regulatory molecules bind non-covalently at the allosteric site. Two types of effectors: i-Positive effectors which stimulate the catalytic reaction ii-Negative effectors inhibit the catalytic reaction The binding of the effector to the allosteric (regulatory) site causes conformational changes in enzyme molecule affecting the active site. Effectors may be the end product of the metabolic pathway, if it inhibits the enzyme of the first reaction, it is called feedback inhibition The Michaelis-Menten equation does not apply and the kinetic curve is sigmoidal shape (not hyperbolic) 26  Enzymes Example: The glycolytic enzyme phosphofructokinase1 is allosterically inhibited by citrate 3- Feedback inhibition: In allosteric regulation in which end products inhibit the activity of the enzyme is called” feedback inhibition”. The end product D thus acts as negative allosteric effector or feedback inhibitor of E1. Example of feedback inhibition: 5- Feedback regulation: It means that the end product of a series of reactions has no inhibitory effect on the first enzyme, but it inhibits the gene needed for the synthesis of first enzyme. 5- Reversible Covalent Modification: By addition of or removal of phosphate, certain enzymes are reversibly activated or inactivated according to body requirements. The phosphate group combines to hydroxyl group of serine and threonine residues of enzyme -Phosphoryation reactions are catalyzed by a family of enzymes called protein kinases that use ATP as a phosphate donor (ATP → ADP + Pi) Dephosphorylation reactions are catalyzed by a family of enzymes called phosphotase. 27  Enzymes Plasma Enzymes: Plasma enzymes are classified into two classes: 1)Functional plasma enzymes which are found in high concentration in plasma and can perform specific function in plasma 2)Non-Functional plasma enzymes which are found in high concentration inside the cells and have a specific function inside the cell but have no function in plasma. Functional Plasma Enzymes: Non-Functional Plasma Enzymes Present in plasma in higher Present in plasma in lower concentrations than in concentrations than in tissues tissues Their substrates are present in blood Their substrates are absent from blood Have known functions No known functions in plasma Mostly synthesized in liver Synthesized in liver, heart, skeletal muscles, brain, Usually decreased in case of Usually elevated in diseased conditions such as cell diseased conditions damage Examples: clotting enzymes, Examples: AST, ALT, CPK, LDH, ACP, ALP, amylase thrombin, fibrinogen, lipoprotein lipase, pseudo-choline esterase Isoenzymes: Isoenzymes (also known as isozymes) are different forms of the same enzymes that differ in amino acid sequence but catalyze the same chemical reaction, act on the same substrate and use the same coenzyme. 28  Enzymes Various isoenzymes of an enzyme can differ in three major ways: 1- chemical properties 2- physical properties (e.g heat stability) 3- Km and Vmax 4- Electrophoretic mobilities 5- Organ distribution 6- biochemical properties such as amino acid composition and immunological reactivity Sources of isoenzymes: 1-Isoenzymes may be synthesized from one gene and processed post-translation. 2- Isoenzyme may be synthesized from more than one gene, where each gene produce one polypeptide chain Example (1): LDH (Lactate dehydrogenase) exists in five different forms each having four polypeptide chains. H= Heart and M=Muscle Diagnostic importance of lactate dehydrogenase isoenzymes: 1- LD1 is elevated in heart diseases (myocardial infarction) 2- LD5 is elevated in liver diseases (infective hepatitis) Example (2): CPK (Creatine phospho kinase) or creatine kinase exists in three different forms each having two polypeptide chains. Characteristic sub-units are B=Brain and M= Muscle. 29  Enzymes Types of enzymes of clinical importance: 1. Transaminases (ALT and AST): - Theses enzymes are present in most tissues, but especially in cardiac muscle and liver. a) ALT activity: is widely used as a test for diagnosis of hepatocellular damage e.g. acute viral hepatitis. b) AST activity: 1) It is used for diagnosis of hepatocellular damage 2) It is increased in myocardial infarction. It reaches its maximum level after 2 day of attack. 2. Alkaline phosphatase a) It shows its maximum activity at pH = 9-10.5 b) It is present in liver, bone, placenta and intestine. c) It is elevated in the following conditions 1) Physiological increase occurs in growing children (bone isomer) and pregnant women (placental isomer) 2) Pathological increase occurs in rickets and hyperparathyroidism (bone isomer) and in obstructive jaundice (liver isomer). 3. Acid phosphatase: a) It shows its maximum activity at pH = 4-5 b) The prostatic acid phosphatase isomer is present in high concentrations in the prostate. c) In prostatic carcinoma PACP is elevated in blood. 4. Lactate dehydrogenase (LD): a) It is present in most tissues especially liver, heart and muscles. b) Its activity is increased in hepatitis, myocardial infarction and muscles diseases. c) In myocardial infarction LD reaches to maximum level after 5 days and returns to its normal level after 5-7 days. 5. Amylase: a) It is produced by pancreas and parotid glands. b) Its activity is elevated in plasma in acute pancreatitis and parotitis. 6. Lipase: a) It is produced by pancreas b) Its activity is elevated in plasma in acute pancreatitis and pancreatic carcinoma. 30  Enzymes 7. Creatine kinase (CK): a) It is also called creatine phosphokinase (CPK) b) It is increase in myocardial infarction and in myopathies. c) In myocardial infarction CK gets its maximum level after 24 hours. d) It returns to its normal level within 2-3 days of attack. 8. Cholinesterase (ChE): a) There are two types of cholinesterase: 1) Plasma cholinesterase is also called pseudocholinesterase, which hydrolyses succinyl dicholine. 2) Tissue cholinesterase is also called true cholinesterase, which hydrolyses acetyl choline. b) Succinyl choline apnea: Some patients during anesthesia and after administration of succinyl dicholine as a muscle relaxant develop prolonged apnea, often lasting for several hours. The plasma of these patients is usually deficient in pseudocholinesterase. 9. Gamma-glutamyl transferase (GGT): a) It is also called gamma-glutamyl transpeptidase. b) It is present in a number of tissues especially kidney and liver. c) Its activity is elevated in plasma in cholestasis (↓ bile flow) and in 70-80% of chronic alcoholics. 31