Biochem 1 Enzymes Lecture Notes PDF

Dr. Mohamed Elsafty Lecturer of Biochemistry Faculty of Dentistry sinaiuniversity.net  Enzymes Lecture 1 37 slides INDEX [email protected] [email protected]...

Dr. Mohamed Elsafty Lecturer of Biochemistry Faculty of Dentistry sinaiuniversity.net  Enzymes Lecture 1 37 slides INDEX [email protected] [email protected] www.su.edu.eg  ILOs Illustrate enzyme, substrate definition, distribution and features. Explain holoenzyme, apoenzyme, prosthetic group and zymogen. Discuss the enzymes terminology and nomenclature. Classify the enzyme specificity. Classify the enzyme classes according to IUB. Explain the mechanism of enzyme action. [email protected] [email protected] www.su.edu.eg  ILOs Clarify the chemistry of active site and theories explain the specificity of enzyme action. List factors affecting enzyme activity. Classify inhibitors and show how they affect enzyme kinetic. Explicate regulation of enzyme activity. Define isoenzymes and describe their clinical significance with examples. Clarify plasma enzyme and enzyme roles in diagnosis, biosensor and therapeutic tool. [email protected] [email protected] www.su.edu.eg ❖ All chemical reactions that occur in living cell have energy barrier separating substrates & products. These barrier are enzymes which aimed to: o Prevent uncontrolled & spontaneous reactions. o Allow reactions to occur at a rate appropriate to the need of the cells. [email protected] [email protected] www.su.edu.eg ❖ Catalysts: are organic & inorganic substances that accelerate the rate of all chemical reactions without being changed or consumed in the reaction. ❖ Organic catalysts: are enzymes. Highly specific: catalyze one or two reactions. Protein in nature, so they are denatured by heat. ❖ Inorganic catalysts: as Zn++, Mg++ ions. Nonspecific: catalyze many reactions. Not affected by heat. [email protected] [email protected] www.su.edu.eg  Enzymes ❖ Enzymes are biocatalysts mainly protein in nature that accelerate or regulate the rate of all biochemical reactions without being changed or consumed in the reaction. ❖ Rate of chemical reaction: It is the change in the amount of substrates or products per unit time. ❖ Substrate: The substance acted upon by enzyme [email protected] [email protected] www.su.edu.eg Cellular distribution of enzymes a) Intracellular: produced and act inside the cells e.g. metabolic enzymes. b) Extracellular: produced inside the cells and act outside e.g. digestive enzymes. The common features of enzymes: They are protein in nature and are genetically determined. They act with moderate pH & temperature (thermolabile). They accelerate the reactions by decreasing energy of activation. The set of enzymes in each cell is genetically determined. Enzymes are highly specific there are different types. [email protected] [email protected] www.su.edu.eg  Enzyme specificity 1. Optical specificity: act on 2 isomers e.g. maltase act on α glycosides not β. 2. Group specificity: need the presence of certain group to act e.g. pepsin act on peptide bond. PICTURE HERE 3. Absolute specificity: act only on one substrate e.g. urease acts only on urea. 4. Relative specificity: acts on a group of compounds having the same type of bond e.g. lipase act on different triacylglycerols. 5. Reaction specificity: there are 6 enzyme classes.  Enzyme activity Requirements for enzyme activity: enzymes are either 1. Simple protein enzyme: is formed of protein only e.g. lipase, amylase. PICTURE HERE 2. Conjugated protein enzyme: also called holoenzymes consisting of: Apoenzyme: protein part. Cofactor: Non-protein part. [email protected] [email protected] www.su.edu.eg Cofactor: These are substances that are required for an enzyme to be catalytically active. It may be organic or inorganic. It include coenzymes and metal ions. Coenzymes: Organic molecules. Loosely attached to apoenzyme. Usually derived from vitamins. Glutathione is a non-vitamin coenzyme. Metal ions: Inorganic molecules e.g. Zn, Fe+2, Cu+2 and Mg.+2 [email protected] [email protected] www.su.edu.eg Functions of cofactors: Activators of enzymes. Carriers to parts removed from substrates. Donors to parts added to the substrates. [email protected] [email protected] www.su.edu.eg Coenzymes and metals which act as carriers & classified into : I. Hydrogen and electron carriers: they include the following: Nicotinamide derivatives (NAD, NADP) Flavin derivatives (FMN and FAD). CoQ II. Other group carriers: they are vitamin derivatives and include the following: Thiamine pyrophosphate (TPP) & biotin: carry C=O and CO2. Pyridoxal phosphate (PLP): carry NH2. Folic acid (FH4): carry one carbon moiety. Cobalamin: carry methyl group. [email protected] [email protected] www.su.edu.eg  Zymogens They are inactive enzymes because their catalytic sites masked by polypeptide chain. PICTURE HERE To activate zymogen , polypeptide chain should be cleaved to open the catalytic site. [email protected] [email protected] www.su.edu.eg  Enzyme Terminology Some enzymes have been retained its old name (trivial) e.g. trypsin, pepsin. Others named by adding the suffix ase to the name of substrate e.g., maltase, lactase and sucrase. Some enzymes were named according to the type of the reaction e.g. aminotransferase that transfer amino group. To standardize enzyme nomenclature, (IUB) made a systemic name to each enzyme. This name can indicate: 1. The substrate acted upon. 2. The coenzyme involved in the reaction. 3. The type of reaction catalyzed. e.g. Lactate - NAD+ - oxidoreductase enzyme. (Its old name was lactate dehydrogenase). It catalyzes the following reaction: [email protected] [email protected] www.su.edu.eg  Enzyme commission number (E.C. number ): is a numerical classification scheme for enzymes, based on the chemical reactions they catalyze it contain 4 digits E.C.1.1.1.1. (class. Functional grp. Coenzyme. Substrate of coenzyme). e.g. Alcohol-NAD- dehydrogenase: E.c. 1.1.1.1 R·CH2-0H + NAD+ ~ R-CHO + NADH + H+ E.C (1 ): Class of enzyme: oxidoreductase. E.C.1.(1 ): Group upon which the enzyme acts is : CH2-OH. E.C.1.1.(1):The coenzyme is NAD. E.C.1.1.1.(1):Alcohol e.g. ethanol is the substrate [email protected] [email protected] www.su.edu.eg  Classification of enzymes there are six classes of enzymes: 1. Oxidoreductase: These enzyme catalyze oxidation reduction reaction between two substrates by removal of H (dehydrogenase) or by addition of O (oxidase). PICTURE HERE 2. Transferases: These enzyme catalyze transfer of a group other than hydrogen from one substrate to another as phosphotransferase, transaminase& transmethylase. 3. Hydrolases: These enzyme catalyze hydrolysis i.e break down of chemical bond by addition of water e.g. peptidase hydrolyze dipeptide into 2 amino acid. [email protected] [email protected] www.su.edu.eg 4. Lyases: These enzyme catalyze removal of group from substrate by mechanism other than hydrolysis. 5. Isomerases: Enzymes catalyze the interconversion of one isomer into other. PICTURE HERE 6. Ligases: These enzyme catalyze joining of two substrate using the energy release from ATP hydrolysis. [email protected] [email protected] www.su.edu.eg  Mechanism of Enzymes Action Substrates (final state) energy product (final state) Ground state activation energy transitional product PICTURE HERE Activation energy: the amount of energy required to raise all molecule of one mole substrate to transition state. The effect of enzymes is to decrease the activation energy. [email protected] [email protected] www.su.edu.eg  Chemistry of active site Is the region of an enzyme where substrate molecules bind and undergo a chemical reaction forming enzyme-substrate complex followed by dissociation and produce enzyme + product. [email protected] [email protected] www.su.edu.eg Two theories explain the specificity of enzyme action: Lock & key theory: active site of enzyme is complementary in conformation to substrate for binding. PICTURE HERE Induced fit theory: enzyme change shape upon biding the substrate, so the conformation of enzyme& substrate are complementary after binding. [email protected] [email protected] www.su.edu.eg  Factors Affecting Reaction Velocity 1-Temperature Optimum temp. for enzymatic activity is 37 o c. PICTURE HERE 2- pH the optimal pH for enzyme is the pH at which enzyme acts maximally. Below or above the activity decline. Each enzyme has its own optimal pH. [email protected] [email protected] www.su.edu.eg 3. Enzyme concentration: The velocity of the reaction increases as the enzyme concentration increases up to certain point. Above this point any increase in the enzyme concentration will not increase in reaction because the substrate is completely utilized. PICTURE HERE 4. Substrate concentration: The velocity of the reaction is directly proportional to substrate concentration up to certain point. Above this point any increase in the substrate concentration will not increase in reaction because the active sites are saturated. [email protected] [email protected] www.su.edu.eg 5. Enzyme activators: They include: a) Metal Ions: e.g. chloride ions activate salivary amylase and Ca++ activate blood clotting enzymes. b) Enzymes: Some inactive enzymes (zymogens) may need other enzymes for activation. This includes: 1) Autoactivation: e.g. pepsinogen to Pepsin by pepsin. 2) Other enzymes: e.g. trypsinogen to trypsin by Enteropeptidase. c) HCI: It starts activation of pepsinogen into pepsin. d) Bile slats: Activate pancreatic lipase enzyme. [email protected] [email protected] www.su.edu.eg  Enzyme Inhibitors These are substances that diminish the velocity of enzymatic reactions. Types: A. Reversible inhibitors include: Bind to enzymes through noncovalent bond and the enzyme activity can be recovered. 1. Competitive 2. Noncompetitive B. Irreversible inhibitors This inhibition cannot be reverse, and the inhibitor alter the active site prevent enzyme substrate binding. [email protected] [email protected] www.su.edu.eg  A. Reversible inhibitors 1. Competitive inhibition: The inhibitor and substrate are similar but not identical in the structure, so both can bind the catalytic site of the enzyme. The degree of inhibition depends on the relative concentration of both and the inhibition can be reversed by increasing the concentration of substrate. [email protected] [email protected] www.su.edu.eg Examples: 1. Allopurinol: this drug is used in treatment of gout. Allopurinol has structural similarity to hypoxanthine. It inhibits xanthine oxidase that oxidizes hypoxanthine to xanthine then to uric acid. PICTURE HERE 2. Dicoumarol and warfarin: act as anticoagulants. They are structurally similar to vitamin K. [email protected] [email protected] www.su.edu.eg 3. Statin drugs: Such as atorvastatin (Lipitor) and simvastatin (Zocor) are structural analogs of the natural substrate for beta- hydroxylmethylglutaryl CoA reductase (HMG-CoA Reductase), the key enzyme of cholesterol synthesis. PICTURE HERE 4. Sulfanilamide and p-aminobenzoic acid (PABA): PABA is required for synthesis of folic acid in bacteria. Folic acid is essential for bacterial growth and multiplication. Sulfanilamide acts as competitive inhibitor for enzyme system that uses PABA. [email protected] [email protected] www.su.edu.eg 2. Non- Competitive inhibition: Inhibitor and substrate bind to different sites on the enzyme. Characters of the combination of enzyme and Inhibitor: a. The inhibitor does not alter the catalytic site (binding). b. No structural similarity between substrate and inhibitor. PICTURE HERE c. The inhibitor can bind either free enzyme or the enzyme- substrate complex. Both enzyme inhibitor complex and enzyme substrate inhibitor complex are inactive. 3· Example of noncompetitive Inhibitors: Allosteric inhibitors & Feed back Inhibitors.  B. Irreversible enzyme inhibitors This type of inhibition cannot be reversed by addition of more substrate. It includes: 1- All factors that produce denaturation or precipitation of proteins: e.g. high temperature, strong acids and alkalis. 2-Inhibitors of the sulfhydryl group: The presence of free SH group is important for the catalytic activity of many enzymes. Inhibitors of the SH group include the following: Oxidizing agents (H2O2), salts of heavy metals (Hg++). [email protected] [email protected] www.su.edu.eg 3-Antienzymes : Antienzyme are specific for the enzymes that binds with it and produces its inactivation e.g. antithrombin and α-1 antitrypsin. 4-Inhibition by removal of catalytic ions: Addition of ethylene Fluoride salts inhibit blood clotting by removal of Ca2+ and inhibit enolase enzyme of glycolysis by removal of Mg2+. e diamine tetra acetic acid (EDTA) to prevent blood clotting by removal of Ca2+. 5-Inhibitors of coenzymes or prosthetic groups: In the case of cyanide or carbon monoxide poisoning, each can bind with iron of hem present in cytochrome oxidase. [email protected] [email protected] www.su.edu.eg  Clinical importance of enzyme inhibitors Antithrombin III: which is activated by heparin and prevents blood clotting. Antiproteinases: Alpha 1-anti-trypsin antagonizes the action of elastase which is released into normal tissues and increased markedly during inflammation. [email protected] [email protected] www.su.edu.eg  Regulation of enzyme activity ❑ Amount of enzyme present: depend on inducer, repressor and adaptation. ❑ Allosteric regulation: PICTURE HERE + or – effector ❑ Feed back inhibition. ❑ Feed back regulation. ❑ Phosphorylation and dephosphorylation. [email protected] [email protected] www.su.edu.eg [email protected] [email protected] www.su.edu.eg  Isoenzymes Isoenzymes: are different forms of enzymes having the same catalytic activity but differs in structure, substrate affinity and tissue distributions. The best examples of isoenzymes are lactate dehydrogenase and creatine kinase isoenzymes [email protected] [email protected] www.su.edu.eg 1-Lactate dehydrogenase (LDH): is composed of 4 polypeptide chains each may be one of two types H and M. There are 5 LDH isoenzymes : LDH1 HHHH present in heart LDH2 HHHM present in heart LDH3 HHMM present in WBCs LDH4 HMMM present in liver LDH5 MMMM present in liver LDH1 and LDH2 are increased in myocardial infarction. LDH5 is increased in liver diseases (hepatitis). [email protected] [email protected] www.su.edu.eg 2-Creatine kinase (CK ): is composed of 2 subunits M and B. CK have 3 isoenzymes : CK BB present in brain CKMB present in heart CK MM present in muscle CK BB: is increased in brain cancer and injury. CKMB: is increased in myocardial infarction, open heart injury ,inflammation of cardiac muscle. CKMM: is increased in muscle damage, or inflammation , intramuscular injection, and strenuous exercise. [email protected] [email protected] www.su.edu.eg  Plasma Enzymes Clinical importance of enzymes : Enzymes are the preferred markers in various disease states such as myocardial infarction, jaundice, pancreatitis, cancer. They provide insight into the disease process by diagnosis, prognosis and assessment of response therapy. The application of enzymes can be broadly classified into: (I) Enzymes in diagnosis. (II) Enzymes in diagnostics–biosensors. (III)Enzymes as a therapeutic tools. [email protected] [email protected] www.su.edu.eg  I- Enzymes in diagnosis Enzyme Disease ALT Liver disease Acid phosphatase Cancer prostate Alkaline phosphatase Bone disease Amylase Pancreatitis [email protected] [email protected] www.su.edu.eg  II- Enzymes in diagnostics-biosensors [email protected] [email protected] www.su.edu.eg  III) Enzymes as a therapeutic tools Enzyme Disease Streptokinase Myocardial infarction Asparaginase Leukaemia Collagenase Skin ulcers [email protected] [email protected] www.su.edu.eg  References Champe PC, Harvey RA, Ferrier DR, Lippincott William & Wilkins. Lippincott Reviews of Biochemistry, London, 7th edition; 2017. https://themedicalbiochemistrypage.org/resources.php [email protected] [email protected] www.su.edu.eg  THANK YOU For any questions feel free to contact me by mail [email protected] 01147996609 Dr. Mohamed Elsafty Lecturer of Biochemistry Faculty of Dentistry