Enzyme Biochemistry Lecture Notes PDF

ENZYMES Dr. Maghawry Hegazy Lecturer of Biochemistry and Molecular Biology Faculty of Pharmacy, Al-Azhar University (Boys), Cairo   History OBJECTIVES  Structure  Mechanism of action  Cofactors and Coenzymes  Classification and nome...

ENZYMES Dr. Maghawry Hegazy Lecturer of Biochemistry and Molecular Biology Faculty of Pharmacy, Al-Azhar University (Boys), Cairo   History OBJECTIVES  Structure  Mechanism of action  Cofactors and Coenzymes  Classification and nomenclature  Specificity  Catalytic efficacy and localization  Factors affecting enzyme activity  Kinetics  Enzyme inhibition  Enzyme regulation  Isozymes  Anti-enzymes  Enzymes in clinical medicine History As early as the late 17th and early 18th centuries, the digestion of meat by stomach secretions and the conversion of starch to sugars by plant extracts and saliva were known. However, the mechanism by which this occurred had not been identified. In the 19th century, when studying the fermentation of sugar to alcohol by yeast by Louis Pasteur called it "ferments". Eduard Buchner in 1897 at the University of Berlin. He named the enzyme "zymase". In 1907, he received the Nobel Prize. In USA, John Howard Northrop and Wendell Meredith Stanley, who worked on the digestive enzymes pepsin (1930), trypsin and chymotrypsin and were awarded the 1946 Nobel Prize in Chemistry. An English group led by David Chilton Phillips ( published in 1965 ) solved enzymes structures by x-ray crystallography. First, for lysozyme. Enzymes Enzymes are biological catalysts, organic thermo-labile, accelerate the reaction without changed. An enzyme's name is often derived from its substrate or the chemical reaction it catalyzes, with the word ending in –ase. Structure of enzymes All enzymes are protein in nature except ribosomes which are RNA in nature Enzymes are usually much larger than their substrates.  Structure of enzymes Complex or holoenzymes (protein part Simple (only protein) and non-protein part – cofactor) Apoenzyme (protein part) Cofactor Coenzyme Prosthetic groups Cofactors: Non-protein compound that is required for an enzyme's activity as a catalyst can be either inorganic (e.g., metal ions and iron-sulfur clusters) or organic compounds (e.g., flavin and heme). Cofactors can be either coenzymes, which are released from the enzyme's active site during the reaction, or prosthetic groups, which are tightly bound to an enzyme. Cofactors can be considered "helper molecules" 7 Coenzymes: Some coenzymes, such as NAD, FMN, FAD, TPP, and THF, are derived from vitamins. These coenzymes cannot be synthesized by the body de novo. Thermo-labile Loosely bound to apoenzyme It is useful to consider coenzymes to be a special class of substrates. Coenzymes are usually continuously regenerated, and their concentrations maintained at a steady level inside the cell. Prosthetic group: Cofactor that is firmly bound to enzyme and cannot be separated without denaturation. Most of prosthetic groups are inorganic and contain an atom as magnesium, cupper and zinc Thermo-stable 8 features of active site 1. The existence of active site is due to the tertiary structure of protein resulting in three-dimensional native conformation. 2. The active site is made up of amino acids (known as catalytic residues) which are far from each other in the linear sequence of amino acids (primary structure of protein). 3. The active site is not rigid in structure and shape. It is rather flexible to promote the specific substrate binding. 4. Generally, the active site possesses a substrate binding site and a catalytic site. The latter is for the catalysis of the specific reaction. 5. The coenzymes or cofactors on which some enzymes depend are present as a part of the catalytic site. 6. The substrate(s) binds at the active site by weak noncovalent bonds. 9 7. Enzymes are specific in their function due to the existence of active sites.  Mechanism of action Substrate binding Enzymes must bind their substrates before they can catalyze any chemical reaction. Enzymes are usually very specific as achieved by binding pockets with complementary shape and charge of the substrates. Enzymes can accelerate reactions in several ways, all of which lower the activation energy ( the minimum amount of extra energy required by a reacting molecule to get converted into product) ## (ΔG‡, Gibbs free energy) 1) Positioning substrates together in the proper orientation 2) Applying torque on the substrates, providing an alternative reaction pathway 3) Providing the proper charge or pH microenvironment 4) Adding or removing functional groups on the substrates  "Lock and key" model 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. This early model explains enzyme specificity, but fails to explain the stabilization of the transition state that enzymes achieve.  Induced fit model Since enzymes are rather flexible structures, the active site is continuously reshaped by interactions with the substrate as the substrate interacts with the enzyme. In some cases, such as glycosidases  Classification and nomenclature Old trivial name Pepsin → Pepsis → Digestion. Lysozymes → Lysis. Recommended (daily used)  Adding ase as a suffix for the substrate → Urease, Lactase, Sucrase..etc.  Substrate/product + action performed + ase → Aspartate Amino Transferase. The International Union of Biochemistry and Molecular Biology have developed a nomenclature for enzymes, the EC numbers (for "Enzyme Commission"). Each enzyme is described by "EC" followed by a sequence of four numbers which represent the hierarchy of enzymatic activity (from very general to very specific). That is, the first number broadly classifies the enzyme based on its mechanism while the other digits add more and more specificity. 1- The first digit denotes reaction type 2- The second digit denotes the functional group 3- The third digit denotes the coenzyme 4- The fourth digit denotes the substrate  The top-level classification is: EC 1, Oxidoreductases catalyze oxidation/reduction reactions EC 2, Transferases transfer a functional group (e.g. a methyl or phosphate group) EC 3, Hydrolases catalyze the hydrolysis of various bonds EC 4, Lyases cleave various bonds by means other than hydrolysis and oxidation EC 5, Isomerases catalyze isomerization changes within a single molecule EC 6, Ligases join two molecules with covalent bonds. EC 7, Translocases catalyze the movement of ions or molecules across membranes. These sections are subdivided by other features such as the substrate, products, and chemical mechanism.  Hexokinase (EC 2.7.1.1) is a transferase (EC 2) that adds a phosphate group (EC 2.7) to a molecule containing an alcohol group (EC 2.7.1), hexose sugar as asubstrate (EC 2.7.1.1). EC categories do not reflect sequence similarity. For instance, two ligases of the same EC number that catalyze exactly the same reaction can have completely different sequences. COMT (EC 2.1.1.6) ……………………..  E.C.1: Oxidoreductases Using NAD/NADH+H, NADP/NADPH+H, FAD/FADH2, FMN/FMNH2 as coenzymes. including reductase, oxidases, hydroxylases, catalases, dehydrogenases, oxygenases and peroxidases 1. Oxidases Catalyze direct transfer of hydrogen to oxygen and form water, cytochrome oxidase (EC 1.9.3.1) 2. Aerobic dehydrogenases Catalyze direct transfer of hydrogen to oxygen and form hydrogen peroxide, amino oxidase (EC 1.4.3.3) 3. Anerobic dehydrogenases Hydrogen indirectly transferred to oxygen through many hydrogen carriers, glucose-6-phosphate dehydrogenase (EC 1.1.1.49) 4. Hydroperoxidases Use hydrogen peroxide as a substrate to form water, peroxidase (EC 1.11.1.7) 5. Oxygenases Catalyze direct incorporation of oxygen into substrate  Mono-oxygenase (hydroxylase), phenyl-alanine hydroxylase (EC 1.14.16.1)  Di-oxygenase, tryptophan pyrrolase (EC 1.13.11.11)  (EC 1.4.3.2) (EC 1.8.1.7) (EC 1.11.1.9) (EC 1.11.1.6) 19  Usually having Trans or Transferase segment in their names, with an exception for kinases (Phosphorylating enzymes). Using donors according to their corresponding reactions: Acetyl COA → Acetylation. SAM → Methylation ATP → Phosphorylation (Kinases) 1. Transaminases Catalyze transfer of amino group NH2 from amino acid to α-keto acid, glutamic-pyruvic transaminase (EC 2.6.1.2) 2. Acyl transferases Catalyze transfer of acyl (fatty acid), need Co-A as a carrier for acyl group, choline acetylase (EC 2.3.1.6) 3. Methyl transferases Catalyze transfer of methyl group using SAM as a donor, COMT (EC 2.1.1.6) 4. Phosphotransferases Catalyze transfer of phosphate group, glucokinase (EC 2.7.1.2)  (EC 2.1.4.1) (EC 2.1.1.2) (EC 2.1.1.6) (EC 1.4.3.4)  E.C.3: Hydrolases Using H2O for bonds cleavage. Don’t forget HYDRO (H2O) LYSIS (Break down). Peptidases Lipases Glycosidases Phosphatases Ribo and Esterases Amidases Deoxyribonucleases Lysozymes (RNases & DNases) (EC 3.6.1) 22  E.C.4: Lyases Catalyze lysis of a substrate, generating a double bond in a nonhydrolytic, nonoxidative elimination. Ex: Synthases, Decarboxylases, Aldolases..etc.  Aldolase : splits aldehyde from alcohols, fructose 1,6 diphosphate aldolase  Dehydratase : removes water from their substrats, carbonic anhydrase  Phosphorylase : cleavages substrats by addition of phosphoric acid, glycogen phosphorylase  Decarboxylase : removes CO2 from substrats, needs pyridoxal phosphate (PLP) as co- enzyme, histidine decarboxylase (EC 4.1.1.1) E.C.5: Isomerases Catalyze isomerization reaction. Ex: Isomerases. Mutases, racemases and epimerases. 1. Aldose-ketose isomerase Catalyze interconversion between aldoses and ketoses as conversion between glucose-6-phosphate to fructose-6-phosphate 2. Epimerases Catalyze interconversion between epimers as glucose and galactose (phosph-hexose isomerase EC 5.3.1.9) 24 3. Mutases Catalyze transfer of group from one position to another in the same compound, glucose-6- phosphate and glucose-1-phosphate 4. Cis-trans Catalyze interconversion between cis and trans geometric isomers, cis and trans retinol 5. Racemases Catalyze interconversion between D and L enantiomers, D and L alanine (EC 5.4.2.2)  E.C.6: Ligases Catalyze ligation reaction, in presence of ATP. Ex: Synthetase, Carboxylase, DNA ligase. Glutamine synthetase (EC 6.3.1.2) (EC 6.4.1.2)  E.C.6: Translocases Catalyzing the translocation of other compounds Carnitine-acylcarnitine translocase ADP/ATP translocases  Specificity Enzymes are highly specific, enzymes possess most elegant specificity of all the catalysts known. Typically, each individual reaction requires its own enzyme, and if an enzyme is lacking, only one particular reaction is generally blocked. Stereo-specificity (Optical) Enzyme acts on one of 2 isomers such as amylase enzyme acts on α 1,4 glycosidic linkage not β-one. Relative broad specificity One enzyme acts on group of compounds having the same type of bonds Pepsin → Peptide bonds. Lipases → Triacylglycerols. Hexokinases → Glucose, fructose and mannose.  Catalytic efficiency Catalytic efficiency of enzymes is very high, most of them bring about several-fold (103 to 1015 times) increase in the reaction rate. Typically, each enzyme molecule is capable of transforming 100 to 1000 substrate molecules into the corresponding product molecules each second. Carbonic anhydrase, the enzyme catalyzing formation of carbonic acid from water and carbon dioxide, is highly active; a single enzyme molecule can transform 36,000,000 substrate molecules each second. 29 Localization Enzymes are sometimes considered under two broad categories: (a) Intracellular enzymes –They are functional within cells where they are synthesized. (b) Extracellular enzymes – These enzymes are active outside the cell; all the digestive enzymes belong to this group. Such arrangement helps to: Organize thousands of enzymes of the cell in distinct pathways. Provide favorable environment for cellular reactions. Isolate the substrate or product of a given reaction from other competing reactions. 1- Effect of enzyme concentration  The rate of enzyme action is directly proportional to the concentration of enzyme provided that there are sufficient supply of substrate & constant conditions.  2- Effect of substrate concentration  The rate of reaction increases as the substrate concentration increases up to certain point at which the reaction rate is maximal (Vmax).  At Vmax, the enzyme is completely saturated with the substrate, any increase in substrate concentration does not affect the reaction rate. 3- Effect of temperature  Rate of reaction increases gradually with the rise in temperature until reach a maximum at a certain temperature, called optimum temperature.  The optimum temperature is 37- 39 °C in humans.  4- Effect of pH  Each enzyme has an optimum pH at which its activity is maximal. Examples : Optimum pH of pepsin =1.5-2 Optimum pH of pancreatic lipase = 7.5-8 Optimum pH of salivary amylase = 6.8 5- Concentration of coenzymes In the conjugated enzymes that need coenzymes, the increase in the coenzyme concentration will increase the reaction rate. 6- Concentration of ion activators Increase in metal ion activator increase the reaction rate as 1- Chloride ion activate salivary amylase. 2- calcium ion activate thrombokinase enzyme. 7- Effect of time  In an enzymatic reaction, the rate of reaction is decreased by time.  This is due to: 1- the decrease in substrate concentration. 2- The accumulation of the end products. 3- The change in pH than optimum pH. 8- Presence of enzyme inhibitors  Presence of enzyme inhibitor decreases or stops the enzyme activity.  Kinetics The study of the rate at which an enzyme acts. Vmax is the reaction rate when the enzyme is fully saturated by substrate. Michaelis–Menten constant (Km) is the substrate concentration required for an enzyme to reach one-half its maximum reaction rate; generally, each enzyme has a characteristic Km for a given substrate. Turnover number ( kcat ) , which is the number of substrate molecules handled by one active site per second. Enzymes with low Km: have high affinity to the substrate i.e. they act at maximal velocity at low substrate concentration. E.g. Hexokinase acts on glucose at low concentration (fasting state). Enzymes with high Km: they have low affinity to the substrate i.e. they act at maximal velocity at high substrate concentration. E.g. Glucokinase enzyme acts on glucose at high concentration (fed state).  Michaelis-Menten hyperbolic plot Enzyme kinetics are studied by plotting the initial velocity (V0) on the Y axis and the substrate concentration (S) on the X axis. The produced curve attains the shape of a rectangular hyperbola. So the curve is called Michaelis- Menten hyperbolic curve. It is hard to draw accurately and hard to determine Vmax and Km precisely.  Vmax is determined by the point where the line crosses the Y axis the (S) is infinite at the point. Km is easily determined from the intercept on the X axis. The curve is linear, so it is widely used as it is easy to draw and provides a more precise way to determine Vmax and Km.  Enzyme inhibition Enzyme inhibition means decreasing or cessation in the enzyme activity. Most therapeutic drugs function by inhibition of a specific enzyme. In the body, some of the processes controlled by enzyme inhibition are blood coagulation, blood clot dissolution (fibrinolysis) and inflammatory reactions. The inhibitor is the substance that decreases or abolishes the rate of enzyme action. According to the similarity between the inhibitor and the substrate, enzyme inhibition is either: 1- Competitive inhibition (Reversible) 2- Non- competitive inhibition (Irreversible) 3- Uncompetitive inhibition (Irreversible) 1- Competitive inhibition There is structural similarity between the inhibitor and substrate. The inhibitor and substrate compete with each other to bind to the same catalytic site of the enzyme. The inhibition is reversible and can be relieved by increasing substrate concentration It does not affect Vmax and increases Km.  Methotrexate is a competitive inhibitor of the enzyme dihydrofolate reductase, which catalyzes the reduction of dihydrofolate to tetrahydrofolate to form dTTP.  Sulfonamides are bacteriostatic by inhibiting dihydropteroate synthetase due to structural similarity to p-amino benzoic that used by bacteria for folic acid synthesis 2- Non-competitive inhibition A- Non-specific non-competitive inhibition As enzymes are proteins in nature, any factor that causes protein denaturation will inhibit enzyme activity e.g. 1- Strong acids 2- Strong alkali 3- Sever agitation 4- Repeated freezing and thawing B- Specific non-competitive inhibition There is no structural similarity between the inhibitor and the substrate. The inhibitor does not bind to the catalytic site but it binds to another site. The substrate still binds with its usual affinity and hence Km remains the same It can bind enzyme or to enzyme substrate complex. The inhibition is irreversible. It cannot be relieved by increasing substrate concentration. It decreases Vmax. It does not affect Km.  Functions of inhibitors As part of a feedback mechanism. Enzymes used as drugs. Methotrexate, sulfonamides and aspirin. The poison cyanide is an irreversible enzyme inhibitor that combines with the copper and iron in the active site of the enzyme cytochrome c oxidase and blocks cellular respiration. The organophosphate pesticides such as malathion, parathion, and chlorpyrifos are enzyme inhibitors. Acetylcholinesterase (AChE).  Peptidoglycan and antibiotics such as penicillin and vancomycin.  Sildenafil (Viagra) is inhibitor of cGMP specific phosphodiesterase type 5 Allosteric regulation of the glycolytic pathway. A key step is phosphofructokinase-1 (PFK1). is a substance that binds to a site on an enzyme or receptor distinct from the active site, resulting in a conformational change that alters the protein's activity, either enhancing or inhibiting its function  Enzyme Regulation Regulation of enzyme activity is important to coordinate the different metabolic processes. It is also important for homeostasis i.e. to maintain the internal environment of the organism constant. it can be achieved by two general mechanisms A- Control of enzyme quantity 1- Control the rate of enzyme synthesis and degradation  Enzymes are protein in nature, they are synthesized from amino acids under gene control and degraded again to amino acids.  Enzyme quantity depends on the rate of enzyme synthesis and the rate of its degradation. Increased enzyme quantity may be due to an increase in the rate of synthesis, a decrease in the rate of degradation or both. Decreased enzyme quantity may be due to a decrease in the rate of synthesis, an increase in the rate of degradation or both.  For example, the quantity of liver Arginase enzyme increases after protein rich meal due to an increase in the rate of its synthesis, also it increases in starved animals due to a decrease the rate of its degradation. 2- Induction  Induction means an increase in the rate of enzyme synthesis by substances called inducers.  According to the response to inducers, enzymes are classified into: 1) Constitutive enzymes, the concentration of these enzymes does not depend on inducers. 2) Inducible enzymes, the concentration of these enzymes depend on the response of inducers.  For example, induction of lactase enzyme in bacteria growth on glucose media.  Omeprazole and lansoprazole are inducers of CYP1A1 and CYP1A2 enzymes through aryl hydrocarbon receptor (AhR), 3- Repression Repression means a decrease in the rate of enzyme synthesis by substances called repressors. Repressors are low molecular weight substances that decrease the rate of enzyme synthesis at the level of gene expression. Repressors are usually end products of biosynthetic reaction, so repression is sometimes called feedback regulation. For example, dietary cholesterol decreases the rate of synthesis of HMG CoA reductase (β- hydroxyl methyl glutaryl CoA reductase), which is a key enzyme in cholesterol synthesis. 4 - Concentration of substrates, coenzymes and metal ion activator The susceptibility of enzyme to degradation depends on its conformation. Presence of substrate, coenzyme or metal ion activator causes changes in the enzyme conformation (leading to decreasing its rate of degradation). B- Control the catalytic efficiency of the enzyme 1- Allosteric regulation  Allosteric enzyme is formed of more than one protein subunit.  It has two sites, a catalytic site for substrate binding and another site (allosteric site), that is the regulation site to which an effector binds. 2- Feedback inhibition In biosynthetic pathways, an end product may directly inhibit an enzyme early in the pathway. This enzyme catalyzes the early functionally irreversible step specific to a particular biosynthetic pathway. Feedback inhibition may occur by simple feedback loop. Negative feedback mechanism can effectively adjust the rate of synthesis of intermediate metabolites according to the demands of the cells. 3- Proenzymes (zymogens) Some enzymes are secreted in inactive forms called proenzymes or zymogens. Examples for zymogens include: Pepsinogen, Trypsinogen, Chemotrysinogen, prothrombin and clotting factors Zymogen is inactive because it contains an additional polypeptide chain that masks (blocks) the active site of the enzyme. Activation of zymogen occurs by removal of the polypeptide chain that masks the active site. Chymotrypsin, a digestive protease, is produced in inactive form as chymotrypsinogen (proenzyme ) in the pancreas and transported in this form to the stomach where it is activated. This stops the enzyme from digesting the pancreas or other tissues before it enters the gut. 4- Covalent modification It means modification of enzyme activity through formation of covalent bonds e.g. Methylation ( addition of methyl group). Hydroxylation ( addition of hydroxyl group). Phosphorylation (addition of phosphate group).  Isoenzymes Isoenzymes (isozymes) are multiple forms of the enzyme that have the same catalytic activity. Although they have the same catalytic activity, they are physically distinct and differ in electrophoretic mobility and liability to inhibitors. Isoenzyme means the same enzyme. LDH has 5 isoenzymes: LDH1 ( HHHH) it increases in Myocardial infarction. LDH2 ( HHHM) it increases in Myocardial infarction. LDH3 ( HHMM) it increases in Leukemia. LDH4 ( HMMM) it increases in Viral hepatitis. LDH5 ( MMMM) it increases in Viral hepatitis. CK has 3 isoenzymes: CK BB which increases in brain tumors. CK MB which increases in heart diseases. CK MM which increases in skeletal muscle diseases.  Antienzymes These are substances secreted by living cells or organisms that inhibit enzyme activity e.g. Ascaris worms living in the intestine secrete antienzymes (anti-trypsin and anti-pepsin) so, they are not digested by proteolytic enzymes present in the digestive juices. Mucin lining the stomach contains antienzyme (anti-pepsin) that prevents digestion of stomach wall by pepsin. Blood plasms contains natural antienzyme (anti-thrombin) that inactivates thrombin after blood coagulation to prevent its intra-vascular spreading.  Enzymes in clinical medicine The measurement of the serum levels of numerous enzymes has been shown to be of diagnostic significance. This is because the presence of these enzymes in the serum indicates that tissue or cellular damage has occurred resulting in the release of intracellular components into the blood. Alkaline phosphatase: increases in bone and liver diseases. Creatine kinase: is associated with myocardial infarction and muscle diseases. The Creatine kinase are such sensitive indicators of muscle damage. Lactate dehydrogenases(LDH): involved in myocardial infarction, haemolysis and liver disease. Amylase: catalyze the hydrolysis of complex carbohydrates, e.g., glycogen at the -1-4 linkages. The products of this action are maltose and limit dextrins. Amylase is used as a diagnostic aid for pancreatitis.  Alanine aminotransferase (ALT):(GPT), it is sensitive liver indicator. Aspartate aminotransferase (AST): (GOT), is associated with myocardial, hepatic parenchymal and muscle diseases. Glutamyltransferase: This is a carboxypeptidase involved in hepatobiliary disease and alcoholism. Lipase: is involved in pancreatitis and hepatobiliary disease.  Acid phosphatase (ACP), found in prostate and erythrocytes and are used in diagnosis of prostate carcinoma. Glutathione peroxidases: metalloenzymes containing four atoms of selenium per molecule of enzyme, catalyze the oxidation of reduced glutathione by peroxide to form water and oxidized glutathione.