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- 1. Handout of Medical Biochemistry for Clinical Pharmacy Students (I) Level 4 Enzymology By Prof. Manal A. M. Mandour
- 2. Introduction Biochemistry, sometimes called biological chemistry, is the study of chemical processes in living organisms. Biochemistry is a science, whose concern is with vital processes, and which has availed itself of the cell theory and of the principle of the infinite divisibility of matter. By controlling information flow through biochemical signaling and the flow of chemical energy through metabolism, biochemical processes give rise to the complexity of life. Each part of every living being is biochemically connected and 1- provides new ideas and experiments, essential for understanding how life works; 2- supports our understanding of health and disease; 3- drives the discovery of new ways to use molecular systems and their biological functions; 4- and contributes essential innovative information to the technology revolution Much of biochemistry deals with the structures, functions and interactions of cellular components such as proteins, carbohydrates, lipids, nucleic acids and other biomolecules —although increasingly processes rather than individual molecules are the main focus. Among the vast number of different biomolecules, many are complex and large molecules (called biopolymers), which are composed of similar repeating subunits (called monomers). Each class of polymeric biomolecule has a different set of subunit types. For example, a protein is a polymer whose subunits are selected from a set of 20 or more amino acids. Biochemistry studies the chemical properties of important biological molecules, like proteins, and in particular the chemistry of enzyme-catalyzed reactions.
- 3. The biochemistry of cell metabolism and the endocrine system has been extensively described. Other areas of biochemistry include the genetic code (DNA, RNA), protein synthesis, cell membrane transport and signal transduction. Biochemistry is at the heart of life science. It is a fascinating, diverse and sprawling discipline; which makes it near impossible to pigeon-hole or define concisely. Many look upon biochemistry as a science that underpins and explains the essential processes of life, impacting on: Biotechnology and bioinformatics Cell biology and signalling Development and disease Energy and metabolism Genetics Molecular biology Plant biology
- 4. Enzymology Lecture 1 By Prof. Manal A. M. Mandour Medical Biochemistry Department Faculty of Medicine Assiut University
- 5. Intended learning outcomes To clarify and discuss the following: -Introduction. -Definition. -Nature and properties of enzymes. -Systems of enzyme nomenclature. -Classification of Enzymes.
- 6. Introduction -Catalyst is a substance, which accelerates the rate of a chemical reaction without being consumed in the reaction, or affects the end point. -Inorganic catalysts, e.g. copper have the following differences with the organic catalysts (enzymes). Enzymes Inorganic catalysts 1. Thermolabile 1. Thermostable 2. Organic, biologically substances 2. Inorganic, non-biologically substances 3. Protein in nature, denaturable. 3. Non-protein, non-denaturable. 4. Different grades of specificity. 4. Non-specific 5. High catalytic efficiency. 5. Low catalytic efficiency. 6 .Optimum body temp. & pressure. 6. Require high temp. & pressure.
- 7. - Enzyme; en = in, zyme = yeast. Definition: Enzymes are biological catalysts that enable the cell to carryout its chemical activities with maximum speed and efficiency under conditions that are compatible with life……………They are thermolabile organic colloidal catalysts of protein nature produced by the living cells for a specific function of catalyzing chemical reactions. Almost all of the chemical reactions occurring in the body need the presence of certain enzymes. Substrate: It is a substance or substances upon which an enzyme acts, e.g. sucrose is substrate for sucrase enzyme that converts it into glucose and fructose. (Sucrase) Invertase Sucrose ================= Glucose + fructose
- 8. Nature and properties of enzymes 1-They are protein (Globular) in nature and are synthesized on the basis of the genetic information. They have the same properties as proteins, e.g., Denaturation precipitation, electrophoresis, etc. 2-Enzymes are highly specific in their action due to their conformation and can be changed and inactivated (denatured) by effect of temperature, pH, heavy metals organic solvents, and concentrated salt solutions ….etc. Carboxypeptidase Chemotrypsin
- 9. 3-Enzymes form transient reversible complexes with substrates and are not consumed during the reaction. 4-The site on enzyme at which the substrates combine are known as active site binding site, substrate site, or catalytic site. There also sites called allosteric sites. 5- Some enzymes require the help of certain non-protein factors that may be loosely (coenzyme or cofactor) or firmly (prosthetic group) attached to the protein part of the enzyme (apoenzyme). This type of enzymes belongs to conjugated proteins. Holoenzyme = Apoenzyme + Coenzyme and/ or cofactor or prosthetic group. 6-Some enzymes are produced in the form of an inactive form (precursor) called proenzyme or zymogen and they activated by other factors, e.g. pepsinogen is the zymogen of pepsin. + substrate Allosteric site Allosteric inhibitor + Allosteric enzyme Catalytic/Substrate-binding site Conformational Change Substrate binding site unfit No substrate binding, No product + substrate Allosteric activator + Conformational Change Perfect binding Products + Allosteric enzyme
- 10. Citrate synthase enzyme(Globular protein) It condenses Acetyl-CoA & Oxaloacetate to form Citric acid (Citrate)…….This is the first reaction of Krebs Cycle. C CH2 CoASH C ~ SCoA CH3 O Active acetate (Acetyl-CoA) COOH O COOH Oxaloacetate + C CH2 COOH COOH Citrate HO CH2 COOH Citrate sythase H2O
- 11. 7-Some enzymes have isomers and called isoenzymes (isozymes). They are group of enzymes catalyze the same chemical reaction, but differ in some chemical or physical properties. 8- Enzymes may act inside the cells as metabolic enzymes e.g. glycolytic enzymes in cytoplasm, Krebs' cycle enzymes in mitochondria, replication and transcription enzymes in nucleus. They may act outside cells (e.g., the digestive enzymes and blood clotting factors). Lactate dehydrogenase isoenzymes during electrophoretic mobility toward anode (+ve) and separation of the main five types. 1 2 3 4 5 Myocardial infarct Normal Liver disease
- 12. Systems of enzyme nomenclature Substrate-dependent naming: The name is derived from the substrate with -ase as a suffix, e.g., urease (for urea), lipase (for lipids) and protease (for protein). Chemical nature of the reaction-dependent naming: The name depends on nature of chemical reaction with -ase as a suffix, e.g. hydrolase (for hydrolysis) and dehydrogenase (for removal of hydrogen). Both systems , 1&2 are recommended for naming enzymes. Systematic naming: The International Biochemical Union have subdivided enzymes into classes subclasses and sub-subclasses and give every enzyme a written name and a code digital name. The written name is formed of the substrate name, the coenzyme name and name of the chemical process. The digital name is of four digits, the first refers to the enzyme class, the second refers to the subclass, the third refers to the sub-subclass and the fourth refers to the name of the individual enzyme itself. Example is alcohol: NAD: oxidoreductase (1:1:1:1). According to this there are 6 classes of enzymes.
- 13. Classification of Enzymes - Enzymes are classified into 6 classes as follows: Class 1- Oxidoreductases: These are enzymes catalyzing oxidation-reduction reactions e.g., oxidases, oxygenases, reductases, dehydrogenases and peroxidases. Class 2- Transferases: These are enzymes catalyzing the transfer of a chemical group from one compound to the other, e.g. aminotransferases, glycosyltransferases methyltransferases, acyltransferases, phosphotransferases(kinases), transaldolases and transketolases. Class 3- Hydrolases: These are enzymes catalyzing the process of hydrolysis (breakdown of the compound by addition of water), e.g. thiolases, hydrolytic deaminases phospholipases, glycosidases, esterase and peptidases. Class 4- Lyases: - These are enzymes, which catalyze breakdown of substrates by mechanisms other than hydrolysis and oxidation and may leave double bonds. - They include desulfhydrases and dehydratases which reversibly remove or add H2S or water from substrate, An example is fumarase acting on fumaric acid.
- 14. Fumaric acid + H2O Malic acid. - Non-oxidative decarboxylases: which remove or add CO2, e.g., pyruvic decarboxylase, aldolases, lyases or cleavage enzymes are lyases. - Phosphorylases cut the substrate by adding phosphate, e.g., glycogen phosphorylase. (Glycogen)n + H3PO4 (Glycogen)n-1 + Glucose-1-phoaphate Class 5- Isomerases: These are enzymes catalyzing isomerization, e.g.: Epimerases, e.g. UDP-Glucose UDP-Galactose. Cis-trans-isomerase, e.g. trans-Retinol cis-Retinol. Mutase, e.g. glucose-6-phosphate glucose-1-phosphate. Racemase, e.g. D- L-Methylmalonyl-CoA. Aldo-keto isomerase, e.g. Glucose-6- phosphate Fructose-6- phosphate. Class 6- Ligases or synthetases: - These are enzymes catalyzing the process of ligation or binding of 2 molecules together in the presence of ATP (synthetase). Fatty acid + CoASH + ATP Acyl-CoA + AMP + PPi. - Carboxylases are ligases.
- 15. Enzymes Lecture 2 By Prof. Manal A. M. Mandour Medical Biochemistry Department Faculty of Medicine Assiut University + + Free Enzyme Enzyme/Substrate Complex Smaller Products Substrate Free Enzyme Catabolic reaction Anabolic reaction Small Substrates Larger Product
- 16. Intended learning outcomes To clarify and discuss the following: -Substrate specificity of enzymes. -Functional (or active) sites in the enzymes system: 1- Catalytic site. 2- Substrate binding-site. 3-Allosteric site. -Mechanism of enzyme action.
- 17. Substrate specificity of enzymes - There are 5 types of substrate specificity, 1-Absolute specificity. 2. Dual specificity. 3.Stereo specificity. 4.Relative specificity. 5. Structural specificity. 1. Absolute Specificity: In this type of specificity, the enzyme acts on only one substrate, e.g., uricase enzyme acts on uric acid, arginase enzyme acts on arginine, urease enzyme acts on urea, carbonic anhydrase enzyme acts on carbonic acid. 2. Dual specificity: - There are 2 types of dual specificity: An enzyme acting on 2 different substrates but catalyzes one type of reaction, e.g., xanthine oxidase acting on hypoxanthine and xanthine causes oxidation of both substrates into uric acid. Hypoxanthine Xanthine Uric acid An enzyme acting on one substrate but catalyzes 2 different reactions, e.g., isocitrate dehydrogenase acts on isocitrate causing dehydrogenation and decarboxylation into -ketoglutarate. Isocitrate + NAD CO2 + NADH.H+ + -ketoglutarate
- 18. 3. Stereo-specificity: In this type of specificity, the enzyme is specific to a specific isomer of a substrate and does not act on other isomers, e.g., L-amino acid oxidase acting on L-amino acids only and D-amino acid oxidase acting on D-amino acids only. All metabolic enzymes act on D-sugars and L-amino acids only. 4. Relative specificity: In this type, the enzyme acts on a group of compounds related to each other in having the same type of bond and also this enzyme catalysis the same type of reaction, e.g., Lipase catalyzes the process of hydrolysis of ester linkage present in triglycerides containing different types of fatty acids. Amylase catalyzes the process of hydrolysis of glycosidic linkages present in starch, dextrin or glycogen. Proteases hydrolyze peptide bonds in different proteins. 5. Structural specificity: - In this type of specificity, the enzyme is specific to the bond like the relative specificity but it requires chemical groups or atoms around this bond. Pepsin hydrolyzes the middle or terminal peptide linkages formed by the amino groups of phenylalanine or tyrosine. Trypsin attacks the peptide linkage containing the carboxyl group of arginine or lysine. - This type of specificity is sometimes described as group specificity such as amino- peptidases and carboxy-peptidase, both break peptide bonds but the first prefer the amino end of the polypeptide chain, whereas, the second type prefer the carboxy end.
- 19. Functional (or active) sites in the enzymes system: a) Catalytic site. b) Substrate binding-site. c) Allosteric site. a) Catalytic site: - It is the region on the enzyme surface that catalyzes the chemical reaction, in other words, it is the site(s) which manipulates the substrate to help rapidity of the chemical reaction. It may be separated from the substrate-binding site by a large or a small distant or they may be combined into one site. b) Substrate-binding site: - Substrate-binding site at which substrate specifically binds and the active site is the site that carries out the chemical action. **The catalytic site and/or substrate binding-site may be rigid or flexible. In the rigid model, they have rigid tertiary structure and does not change their shape after combination with substrate. So the substrate must have a complementary shape and size in order to fit in the catalytic site. This is described as the lock and key model. In the flexible model, the substrate induces a conformational change in the enzyme tertiary structure to fit the substrate. This is described as the induced fitting model.
- 20. Active sites in the enzymes system
- 21. c) Allosteric site: -The term allosteric site means “the other site” and allostery means “a change in shape”. This indicates that when an allosteric effector non-covalently binds at allosteric site (a site other than active and substrate binding site), it causes a conformational change in the enzyme particularity at the active site(s) that decreases or increases the enzyme activity.. -The allosteric site is usually far from the catalytic site(s) at which an allosteric effector binds. Allosteric effectors are substances of low molecular weight having little or no structural similarity to substrate. + substrate Allosteric site Allosteric inhibitor + Allosteric enzyme Catalytic/Substrate-binding site Conformational Change Substrate binding site unfit No substrate binding, No product + substrate Allosteric activator + Conformational Change Perfect binding Products + Allosteric enzyme
- 22. Thus, allosteric effector is called negative allosteric effector (or feedback inhibitor) when the resulting conformational change decreases the enzyme activity. However, the allosteric effector is called positive allosteric effector (or feedback activator) if the resulting conformational change increases the enzyme activity. Mechanism of enzyme action: Enzyme-substrate combination: (see rigid and flexible models) - During the enzyme action, there is a temporary combination between the enzyme and its substrate. Every enzyme has an active site or sites, one of them is the substrate binding site. - The enzyme combines with its substrate to give an enzyme substrate complex. The enzyme then strained or joined bond(s) in the substrate(s) until bond(s) ruptures and gives smaller products (catabolic reaction), or binding small substrates together producing a lager product (anabolic reaction). The enzyme is liberated in a free state to combine with new substrate(s) and so on. So the enzyme acts only as a catalyst for the proceeding of the reaction. + + Free Enzyme Enzyme/Substrate Complex Smaller Products Substrate Free Enzyme Catabolic reaction Anabolic reaction Small Substrates Larger Product
- 23. **Note that:** - Enzymes are proteins have a defined amino acid sequence (100-500 amino acids long). - Enzymes have a defined three-dimensional structure. - They act as a catalyst & increase the speed of the reaction 106-1014 times faster than the rate of the uncatalysed reaction. They speed up rate of reaction by lowering the activation energy. - All enzymes have an active site(s), which contains a small number of catalytic amino acids, which are essential in catalyzing the reaction. - The substrate molecule can bind to the active site via non- covalent interactions (electrostatic interactions, hydrogen bonding, Van der Waals interaction, or hydrophobic interactions.