Enzymes: MLS 2101 Biochemistry for Medical Laboratory Science PDF

Module 7 ENZYMES MLS 2101 Biochemistry for Medical Laboratory Science Melyn J. Pendon, RMT, MSMT │ College of Medical Laboratory Science │ Central Philippine University MLS 2101 MJPendon, RMT, MSMT 1  Learning outcomes At the end o...

Module 7 ENZYMES MLS 2101 Biochemistry for Medical Laboratory Science Melyn J. Pendon, RMT, MSMT │ College of Medical Laboratory Science │ Central Philippine University MLS 2101 MJPendon, RMT, MSMT 1  Learning outcomes At the end of this unit, the students are expected to: 1. Describe the general characteristics and chemical nature of enzymes and its nomenclature. 2. Discuss the factors influencing enzyme activity 3. Identify and describe the six classes of enzymes based on the types of chemical reactions they catalyze 4. Describe the catalytic mechanism and the models of enzyme action. 5. Discuss the enzyme kinetics, the Michaelis-Menten equation & the Lineweaver-Burke Plot. 6. Differentiate the types of enzyme inhibitions and regulation of enzyme activity MLS 2101 MJPendon, RMT, MSMT 2 Course Content A. Classification and nomenclature of enzymes B. Enzyme-substrate binding 1. Lock and key model 2. Induced-fit model C. The Michaelis-Menten Kinetics D. Enzyme inhibition E. Coenzymes and apoproteins F. Enzyme inhibition in the treatment of AIDS MLS 2101 MJPendon, RMT, MSMT 3 General Characteristics of Enzymes Enzyme – is a compound, usually a protein, that acts as a catalyst for a biochemical reaction. Greek word, en-”in” and zyme-”yeast” Catalyst – is a substance that speed up a chemical reaction without itself being consumed during the reaction Enzyme kinetics – is the study of the chemical reactions that are catalyzed by enzymes - The reaction rate is measured and the effects of varying conditions of the reaction are investigated MLS 2101 MJPendon, RMT, MSMT 4  Two General Structural Classes Simple Enzyme are composed only of polypeptide (amino acid chains) it is the tertiary structure of the simple enzymes that makes them biologically active MLS 2101 MJPendon, RMT, MSMT 5  Two General Structural Classes Conjugated Enzyme have a nonprotein part in addition to a protein part a. apoenzyme/apoprotein – protein part of an enzyme, inactive in itself b. cofactor or coenzyme – nonprotein moiety, loosely bound to protein – the activator (biologically active conjugated enzyme) MLS 2101 MJPendon, RMT, MSMT 6  Two General Structural Classes Conjugated Enzyme Two Broad Categories of Cofactors Inorganic (Metal ions) Organic Zinc, Magnesium, Iron ions, Vit. B or B vitamin derivative Copper ions (coenzymes) Coenzyme – is a small organic molecules that serves as a cofactor in a conjugated enzyme MLS 2101 MJPendon, RMT, MSMT 7  Enzyme Nomenclature 1. Suffix -ase identifies as an enzyme e.g., urease, sucrase, and lipase exception: the suffix -in is still found in the names of some digestive enzymes (e.g., trypsin, chymotrypsin, and pepsin), and lysozymes pepsin, from the Greek word pepsis, “digestion” trypsin, named in part from the Greek word tryein, meaning “to wear down” − because it was obtained by rubbing pancreatic tissue with glycerin lysozyme was named for its ability to lyse (break down) bacterial cell walls MLS 2101 MJPendon, RMT, MSMT 8  Enzyme Nomenclature 2. Type of reaction catalyzed by an enzyme is often used with a prefix most enzymes are named with reference to their function oxidase – catalyzes an oxidation reaction hydrolase – catalyzes a hydrolysis reaction 3. Identity of substrate is often used in addition to the type of reaction Substrate – is the reactant in an enzyme-catalyzed reaction. (the substance upon which the enzyme “acts”) glucose oxidase pyruvate carboxylase succinate dehydrogenase Infrequently, the substrate but not the reaction type is given. Urease – catalyzes the hydrolysis of urea lactase- hydrolysis of lactose MLS 2101 MJPendon, RMT, MSMT 9  Enzyme Nomenclature Predicting Enzyme Function from an Enzyme’s Name Predict the function of the following: 1. Cellulase Catalyzes the hydrolysis of cellulose 2. Sucrase Catalyzes the hydrolysis of disaccharide sucrose 3. L-Amino acid oxidase Catalyzes the oxidation of L-amino acids 4. Aspartate aminotransferase Catalyzes the transfer of an -amino group from aspartate to a different molecule MLS 2101 MJPendon, RMT, MSMT 10  Enzyme Commission Number Each enzyme is assigned a four-part classification number and a systematic name, which identifies the reaction it catalyzes. Example: glucose phosphotransferase ATP + D-glucose ADP + D-glucose-6-phosphate Its E.C. number is 2.7.1.1 first number (2) – class name (transferase) second number (7) – subclass (phosphotransferase) third number (1) – a phosphotransferase with a hydroxyl group as acceptor fourth number (1) – D-glucose as the phosphoryl group acceptor MLS 2101 MJPendon, RMT, MSMT 11  Six Major Classes of Enzymes Enzymes are grouped into six major classes based on the types of reactions they catalyze Class Reaction Catalyzed 1. Oxidoreductases Oxidation-reductions 2. Transferases Functional group transfer reactions 3. Hydrolases Hydrolysis reactions 4. Lyases Reactions involving addition of a group to a double bond or removal of groups to form double bonds 5. Isomerases Isomerization reactions 6. Ligases Reactions involving bond formation coupled with ATP hydrolysis MLS 2101 MJPendon, RMT, MSMT 12  Six Major Classes of Enzymes Oxidoreductase An oxidoreductase enzyme catalyzes an oxidation–reduction reaction − an oxidoreductase requires a coenzyme that is either oxidized or reduced as the substrate in the reaction − e.g., lactate dehydrogenase (LDH) is an oxidoreductase that removes hydrogen atoms from a molecule and NAD+ is the coenzyme in this reaction MLS 2101 MJPendon, RMT, MSMT 13  Six Major Classes of Enzymes Transferase a transferase is an enzyme that catalyzes the transfer of a functional group from one molecule to another Two major subtypes: 1. kinases – play a major role in metabolic energy- production reactions - catalyze transfer of a phosphate group from adenosine triphosphate (ATP) to give ADP and a phosphorylated product (contain additional phosphate group) 2. transaminases – catalyze the transfer of an amino group from one molecule to another MLS 2101 MJPendon, RMT, MSMT 14  Six Major Classes of Enzymes Transferase: kinase Hexokinase is an enzyme that catalyzes the phosphorylation of glucose and other hexoses. Phosphorylation of glucose is the process of adding a phosphate group to glucose to create glucose 6-phosphate. (the first step in glycolysis) MLS 2101 MJPendon, RMT, MSMT 15  Six Major Classes of Enzymes Transferase: Transaminase Alanine transaminase (ALT) is an enzyme that catalyzes the transfer of an amino group from alanine to α- ketoglutarate. Pyruvate and glutamate – is the product(reversible transamination) Transamination of alanine to pyruvate allows pyruvate to form glucose through the gluconeogenic pathway MLS 2101 MJPendon, RMT, MSMT 16  Six Major Classes of Enzymes Hydrolase a hydrolase is an enzyme that catalyzes a hydrolysis reaction in which addition of a water molecule to a bond to cause bond to break. hydrolysis reactions are central to the process of digestion a. carbohydrases – hydrolyze glycosidic bonds in oligo- and polysaccharides b. proteases – hydrolyze peptide linkages in proteins c. lipases – hydrolyze ester linkages in triacylglycerols d. Nucleases- hydrolyze sugar-phosphate ester bonds in nucleic acid e. Phosphatases- hydrolyze phosphate-ester bonds MLS 2101 MJPendon, RMT, MSMT 17  Six Major Classes of Enzymes Hydrolase Maltase is an enzyme that catalyzes the hydrolysis of disaccharide maltose to the simple sugar glucose MLS 2101 MJPendon, RMT, MSMT 18  Six Major Classes of Enzymes Hydrolase Triacylglycerol is a primary form of dietary lipid found in fats and oils. Lipase is an enzyme that catalyzes the hydrolysis of TAGs into glycerol and fatty acids MLS 2101 MJPendon, RMT, MSMT 19  Six Major Classes of Enzymes Lyases A lyase is an enzyme that catalyzes the addition of a group to a double bond or the removal of a group in a manner that does not involve hydrolysis or oxidation a. dehydratase – catalyze the removal of the components of water to form a double bond b. hydratase – catalyze the addition of the components of water to a double bond c. decarboxylase – catalyze the removal of carbon dioxide from a substrate d. deaminase – catalyze the removal of ammonia from a substrate MLS 2101 MJPendon, RMT, MSMT 20  Six Major Classes of Enzymes Lyases Fumarate is a metabolic intermediate in the citric acid cycle (TCA). Fumarase catalyzes the reversible hydration and dehydration of fumarate to malate MLS 2101 MJPendon, RMT, MSMT 21  Six Major Classes of Enzymes Isomerase An isomerase is an enzyme that catalyzes the isomerization (rearrangement of atoms) of a substrate in a reaction, converting it into a molecule isomeric with itself. There is only one reactant and one product in the reactions a. racemases – conversion of D- to L-isomer or vice versa b. mutases – transfer of a functional group within a molecule MLS 2101 MJPendon, RMT, MSMT 22  Six Major Classes of Enzymes Isomerase 3- Phosphoglycerate is converted into 2-phosphoglycerate formation via an intramolecular phosphoryl transfer Phosphoglyceromutase relocates the phosphate of the 3- PGA molecules from the 3rd to the 2nd carbon to form 2-PGA molecules MLS 2101 MJPendon, RMT, MSMT 23  Six Major Classes of Enzymes Ligase A ligase is an enzyme that catalyzes the bonding together of two molecules into one involving ATP hydrolysis to ADP ATP hydrolysis is required because such reactions are energetically unfavorable a. synthetases – formation of new bond between two substrates with participation of ATP b. carboxylases – formation of new bond between substrate and carbon dioxide with participation of ATP MLS 2101 MJPendon, RMT, MSMT 24  Six Major Classes of Enzymes Ligase Pyruvate is a product of glycolysis Pyruvate carboxylase catalyzes the carboxylation of pyruvate to oxaloacetate. A key enzyme in gluconeogenesis MLS 2101 MJPendon, RMT, MSMT 25  Main Classes and Subclasses of Enzymes SELECTED MAIN CLASSES TYPES OF REACTION CATALYZED SUBCLASSES Oxidoreductases Oxidases Oxidation of a substrate Reductases Reduction of a substrate Dehydrogenases Introduction of double bond by formal removal of two H atoms from substrate, the H being accepted by a coenzyme Transferases Transaminases Transfer of an amino group between Kinases substrates Transfer of a phosphate group between substrates Hydrolases Lipases Hydrolysis of ester linkages in lipids Proteases Hydrolysis of amide linkages in proteins Nucleases Hydrolysis of sugar-phospahte ester bonds in nucleic acids Carbohydrases Hydrolysis of glycosidic bonds in Phosphatases carbohydrates Hydrolysis of phosphate-ester bonds MLS 2101 MJPendon, RMT, MSMT 26  Main Classes and Subclasses of Enzymes MAIN SELECTED TYPES OF REACTION CATALYZED CLASSES SUBCLASSES Lyases Dehydratases Removal of H2O from a substrate Decarboxylases Removal of CO2 from a substrate Deaminases Removal of NH3 from a substrate Hydratases Addition of H2O to a substrate Isomerases Racemases Conversion of D-isomer to L-isomer, or v.v. Mutases Transfer of a functional group from one position to another in the same molecule Ligases Synthetases Formation of new bond between two substrates, with participation of ATP formation Carboxylases of new bond between a substrate and CO2 with participation of ATP MLS 2101 MJPendon, RMT, MSMT 27  EXERCISE To what main enzyme class do the enzymes that catalyze the following chemical reactions belong? TRANSFERASE LYASE MLS 2101 MJPendon, RMT, MSMT 28  EXERCISE To what main enzyme class do the enzymes that catalyze the following chemical reactions belong? In this reaction: catalyze transfer of a phosphate group from adenosine triphosphate (ATP) to a substrate Enzyme: Transferase (kinase) MLS 2101 MJPendon, RMT, MSMT 29  EXERCISE To what main enzyme class do the enzymes that catalyze the following chemical reactions belong? In this reaction : catalyze the removal of the components of water to form a double bond Lyase (dehydratases) MLS 2101 MJPendon, RMT, MSMT 30  How do Enzyme Work? 1. Binding – The enzymes binds to a substrate molecule at the active site - E attracts S to its active site 2. Stabilizing – The enzyme stabilizes the transition state of the substrate, which speeds up the reaction rate. - catalyzes the chemical reaction 3. Converting – The substrate is converted into product of the reaction. - catalyzes the chemical reaction by which P are formed. 4. Releasing – The product is released from the enzyme - allows the P to dissociate from E surface MLS 2101 MJPendon, RMT, MSMT 31  Substrate and Active Site 1. Substrate (S) – binds to enzyme forming ES complex  the reactant in an enzyme-catalyzed reaction  the substance acted upon by the enzyme  converted into product  e.g., In the fermentation process, sugar is converted to alcohol (in this reaction, sugar is the substrate) E+S ES (Enzyme-Substrate complex) 2. Active site  small portion of the enzyme surface; frequently in a cleft or crevice  optimally orient the substrate to achieve the transition state at a lower energy  essential amino acids are found in or near active site MLS 2101 MJPendon, RMT, MSMT 32  Enzyme Active Site Explanations of how enzymes function as catalysts in biochemical systems are based on the concepts of an enzyme active site and enzyme- substrate complex formation. The active site is relatively small part of an enzyme’s structure that is involved in catalysis − where substrate binds to enzyme − formed due to folding and bending of the protein − usually a “crevice-like” location in the enzyme − some enzymes have more than one active site MLS 2101 MJPendon, RMT, MSMT 33  Enzyme-Substrate Complex Enzymes are highly specific and efficient in their reactions with substrates. They act as catalysts by lowering the activation energy of chemical reactions The substrate initially forms a complex with the enzyme by fitting into active site. The E-S-complex represents the reactive transition state of the reaction, in which the substrate is activated. After subsequent chemical reaction, the product is released, and the enzyme is restored in its original functional conformation MLS 2101 MJPendon, RMT, MSMT 34  Models of Enzyme Action 1. Lock-and-Key model the active site in the enzyme has a fixed, rigid geometrical conformation only substrate of specific shape can bind with active site a substrate whose shape and chemical nature are complementary to those of the active site can interact with the enzyme fails to consider proteins’ conformational changes to accommodate a substrate molecule MLS 2101 MJPendon, RMT, MSMT 35  Models of Enzyme Action 2. Induced-fit Model substrate contact with enzyme will change the shape of the active site allows small change in space to accommodate substrate (e.g., how a hand fits into a glove) the enzyme active site, although not exactly complementary in shape to that of the substrate, is flexible enough that it can adapt to the shape of the substrate MLS 2101 MJPendon, RMT, MSMT 36  Enzyme Specificity Is the extent to which an enzyme’s activity is restricted to specific substrate, group of substrate, chemical bond or type of chemical reaction. The degree is determined by the active site. Some active site can accommodate only one compound. Others accommodate “family” of closely related compounds MLS 2101 MJPendon, RMT, MSMT 37  Enzyme Specificity 1. Absolute specificity an enzyme will catalyze a particular reaction for only one substrate this is most restrictive of all specificities e.g., catalase is an enzyme with absolute specificity for hydrogen peroxide (H2O2) urease absolute specificity for urea 2. Stereochemical specificity an enzyme can distinguish between stereoisomers chirality is inherent in an active site (amino acids are chiral compounds) L-amino-acid oxidase – catalyzes reactions of L-amino acids but not of D-amino acids. MLS 2101 MJPendon, RMT, MSMT 38  Enzyme Specificity 3. Group Specificity involves structurally similar compounds that have the same functional groups e.g., carboxypeptidase cleaves amino acids one at a time from the carboxyl end of the peptide chain 4. Linkage Specificity involves a particular type of bond irrespective of the structural features in the vicinity of the bond considered most general of enzyme specificities e.g., phosphatases hydrolyze phosphate–ester bonds in all types of phosphate esters. MLS 2101 MJPendon, RMT, MSMT 39  Enzyme Activity A measure of the rate at which enzyme converts substrate to products in a biochemical reaction Four factors that affect enzyme activity 1. temperature 2. pH 3. substrate concentration 4. enzyme concentration MLS 2101 MJPendon, RMT, MSMT 40  Enzyme Activity Higher temperature results 1. Temperature in higher kinetic energy which causes an increase in number of reactant collisions, therefore there is higher activity. Optimum temperature is the temperature at which the rate of enzyme-catalyzed reaction is maximum Optimum temperature for human enzymes is 37ºC (body temperature) Increased temperature (high fever) leads to decreased enzyme activity MLS 2101 MJPendon, RMT, MSMT 41  Enzyme Activity optimum pH is the pH at 2. pH which enzyme has maximum activity drastic changes in pH can result in denaturation of proteins most enzymes have optimal activity in the pH range of 7.0 to 7.5 exception: digestive enzymes pepsin: optimum pH = 2.0 chymotrypsin: optimum pH = 8.0 MLS 2101 MJPendon, RMT, MSMT 42  Enzyme Activity H. pylori causes stomach ulcers - It causes more than 90% of duodenal ulcers and up to 80% of gastric ulcers. If digestive system has a pH of 1.0 to 2.5, how does the bacterium H. pylori enzymes with pH of 7.5 to 8.5 able to survive and function in highly acidic environment in the stomach? Urease converts urea = ammonia MLS 2101 MJPendon, RMT, MSMT 43  Enzyme Activity 3. Substrate Concentration At a constant enzyme concentration, the enzyme activity increases with increased substrate concentration. Enzyme saturation is the concentration at which it reaches its maximum rate, and all the active sites are full Turnover number is number of substrate molecules converted to product per second per enzyme molecule under conditions of optimum temperature and pH MLS 2101 MJPendon, RMT, MSMT 44  Enzyme Activity 4. Enzyme Concentration Enzymes are not consumed in the reactions they catalyze At a constant substrate concentration, enzyme activity increases with increase in enzyme concentration − the greater the enzyme concentration, the greater the reaction rate MLS 2101 MJPendon, RMT, MSMT 45 MLS 2101 MJPendon, RMT, MSMT 46  Extremozymes Extremophile - organisms that thrive in extreme environments. Hydrothermophiles thrive at 80-122oC and high pressure Acidophiles optimal growth pH 9.0 Halophiles live in highly saline conditions (>0.2 M NaCl) Piezophiles grow under high hydrostatic pressure (40-110 MPa) Xerophiles grow in extremely dry conditions Cryophiles grow at temps