Enzymes 1, 2, and 3 (Ross University) PDF

Cellular Biology & Homeostasis ENZYMES - Part 1 VP 2024 Clara Camargo, DVM Learning Objectives Define enzyme and the key-lock mechanism. List some industrial applications of enzymes, give some examples. How enzymes work, what is activation energy and the transition state Understand enzyme nomenclatu...

Cellular Biology & Homeostasis ENZYMES - Part 1 VP 2024 Clara Camargo, DVM Learning Objectives Define enzyme and the key-lock mechanism. List some industrial applications of enzymes, give some examples. How enzymes work, what is activation energy and the transition state Understand enzyme nomenclature, give some examples Understand apoenzyme, holoenzyme and cofactors List and explain the properties of the enzymes Enzymes Part 1 RECALL: SUMMARY OF DIGESTIVE ENZYMES Where secreted Salivary glands Stomach Pancreas Small intestine brush border RECAP What enzyme –amylase (Ptyalin) Product of digestion Amylose (polysaccharide) disaccharides Lingual lipase Lipids (TAG, cholesterol) glycerol DAG, MAG, FFA, Pepsin (protease) Proteins Gastric lipase Lipids Pancreatic amylase Polysaccharides Trypsin (protease) Proteins peptides Chymotrypsin (protease) Proteins peptides Acid lipases Lipids Peptidases Polypeptides Nucleotidases, nucleases DNA, RNA Lactase Disaccharides monosaccharides Maltase Disaccharides monosaccharides Sucrase Disaccharides monosaccharides peptides DAG, MAG, FFA, glycerol disaccharides DAG, MAG, FFA, glycerol amino acids nucleotides, ribose RECAP include will most enzymes Most all diseases in animals are manifestations of abnormalities in: biomolecules chemical reactions biochemical pathways Veterinary Physiological Chemistry – Larry R. Engelking ENZYMES General concepts Enzymes are proteins that act as biological catalysts by accelerating chemical reactions. Generally, a small amount of enzyme will The molecules upon which enzymes may act substrate. influence a large amount of reactive are called substrates, and the enzyme converts the substrates into different Act as mediators for virtually all chemical molecules known as products. reactions in biological systems, playing fundamental roles in metabolic events, They have a high degree of specificity for their substrates, and they accelerate chemical reactions tremendously, without being changed or used up during the process (reversible binding). signal transduction and cell regulation. General Enzymes concepts Part 1 ENZYMES The binding is very specific, small changes in the shape of the ligand/substrate (key) can cause major change in protein (lock) behavior. Complementary shape: recognition function, plays a major role in information transfer General Enzymes concepts Part 1 ENZYMES Allostery: the ability of a protein to change shape, resulting in a change in binding affinity at a different binding site. “shape influences binding, and in turn, binding can influence shape” Allosteric enzymes: have the active site, as well as an additional site (allosteric site) From the Greek 'allo', which means 'other'. LIFE DEPENDS ON A COMPLEX NETWORK OF CHEMICAL REACTIONS Almost all metabolic processes in the cell need enzyme catalysis in order to occur at rates fast enough to sustain life. FAST!!! Catalyzed E+S Spontaneous S ES EP SLOW!!! E+P P Binding sites are usually very specific for a particular ligand/substrate and the binding is reversible. Enzymes Part 1 INDUSTRIAL FIELDS ALSO BENEFITS FROM ENZYMES Biofuel production (biodiesel, alcohol from sugar cane) Agricultural (animal feed additives, fertilizers…) Fermentations: transformation of raw materials such as sugar, starch, etc. in industrial mixtures such as liquors, brewing Biotransformations: transformation of defined precursors to a desired target product o Environmentally friendly processes to treat waste Pharmaceutical industry: synthesis & modification of antibiotics & medicines Diagnosis of disease: increased or decreased concentrations of enzyme activity in the target system (liver, kidney, muscle) Treatment of disease: i.e. use of streptokinase to dissolve blood clots, exocrine pancreatic insufficiency… Enzymes Part 1 ENZYMES IN INDUSTRIAL APPLICATIONS Enzymes Part 1 FYI SOME INDUSTRIAL ENZYMES AND THEIR USES Enzyme Application Enzymes Part 1 Sector Protease Degradation of proteins Detergents Cellulase Degradation of cellulose Detergents Lipase Degradation of lipids Detergents Amylase Conversion of starch to glucose Starch processing Glucose Isomerase Production of High Glucose Syrup Starch processing Phytase Improve nutrient availability Animal feed Xylanase Removal of lignin ‘bio bleaching’ Paper and pulp Amylase Removal of fruit starch haze Fruit/vegetable processing Hydrolase Breakers for biopolymer gels Petrol and Gas Chymosin Clotting in cheese manufacture Dairy Pectinase Increased yields Wine HOW ENZYMES WORK? Chemical reactions have an energy barrier separating the reactants and the products. Energy is needed to get them started = Activation energy Enzymes greatly reduce the activation energy barriers that block chemical reactions. Enzymes Part 1 HOW ENZYMES WORK? https://www.youtube.com/watch?v=Dd1yi2aVoOc Enzymes Part 1 HOW ENZYMES WORK? Enzymes direct substrate molecules through a specific reaction pathway allowing a reaction to proceed rapidly by providing an alternate reaction pathway in the cell which has a lower activation energy. Enzymes show a high selectivity and usually catalyze only one specific reaction, or a set of closely related reactions; directing a particular reaction pathway. Enzymes Part 1 Enzymes Part 1 THE TRANSITION STATE The active site acts as a molecular template that binds the substrate and initiates its conversion to the transition state. The transition state is the form the substrate must take before it becomes product. What is: uncat? It is the highest energy point of the reaction. saved infenergy anyway THE TRANSITION STATE Enzymes Part 1 Stabilizing the transition state (T*) an enzyme can greatly increase the concentration of the reactive intermediate that can be converted to product accelerating the reaction. NOMENCLATURE Enzymes Part 1 Recommended name: short name, most used, has the suffix ‘-ase’ attached to the substrate of the reaction: i.e., Glucokinase (found mostly in liver and pancreas, phosphorylation of glucose) the description of the reaction performed: i.e., Lactate dehydrogenase NOMENCLATURE Nomenclature Systematic name: more complete, complex; is used when an enzyme must be identified without ambiguity. The suffix -ase is attached to a more complete description of the chemical reaction catalyzed, including the names of all substrates: LDH (lactate dehydrogenase): Lactate, NAD+oxidoreductase The systematic names are unambiguous and informative, but often too big for general use NOMENCLATURE Enzymes Part 1 Some enzymes retain their original, trivial names, which give no hint of the associated enzymatic reaction. THE MAJOR CLASSES OF ENZYMES Enzymes Part 1 Oxidoreductases: catalyze reactions in which one molecule is oxidized while the other is reduced, transfer of electrons (e-) and hydrogens H+ oxidases, reductases, dehydrogenases, peroxidases + + Classes of Enzymes Transferases: transfer carbon, nitrogen or phosphate groups methyltransferases, aminotransferases, kinases, phosphorylases COO- + COO- + Classes of enzymes Hydrolases: enzymes that catalyze a hydrolytic cleavage reaction (use water to break a chemical bond) *most digestive enzymes are hydrolases nucleases, proteases, phosphatases, amylase, lipase Classes of enzymes Lyases: catalyze the cleavage of C-C, C-S, and C-N bonds (catalyzes the breaking of various chemical bonds by means other than hydrolysis and oxidation, often forming a new double bond or a new ring structure) decarboxylases, aldolases, synthases, polymerases Classes of enzymes Isomerases: catalyze the rearrangement of bonds within a single molecule, transfer of groups within molecules to yield isomeric forms mutases, racemases l =O =O Classes of enzymes Ligases: Join two molecules in an energy-dependent process Catalyze formation of bonds between carbon and O, S, and N coupled to hydrolysis of high energy phosphates Classes of enzymes and Nomenclature Enzymes Part 1 POTENTIALLY CONFUSING ENZYME NOMENCLATURE: Synthetase: requires ATP Synthase: no ATP required Phosphatase: remove phosphates Phosphorylase: add phosphates (cleave bonds by orthophosphate - phosphorolysis) Dehydrogenase: catalyze oxidation/reduction reactions (i.e., Oxidase: O2 is the acceptor of electrons or hydrogen, and oxygen atoms are not incorporated into substrate Oxygenase: catalyze the incorporation of molecular O2 to a substrate. CLASSES OF ENZYMES mostly discussed in our lectures Polymerases: catalyze polymerization reactions such as the synthesis of DNA and RNA Lysases Proteases: break down proteins by hydrolyzing bonds between amino acids Hydrolases Kinases: Catalyze the addition of phosphate groups to molecules (protein kinases are very common in physiology) Transferases ATPases: Hydrolyze ATP (Na, K- ATPase) Synthases: synthesize molecules in anabolic reactions by condensing two smaller Lysases molecules together without using ATP Phosphatase: catalyze the hydrolytic removal of a phosphate group from a molecule Hydrolases PROPERTIES and CHARACTERISTICS OF ENZYMES Active sites: enzymes contain a special pocket called the ‘active site’ which has a high specificity Contains amino acid side chains that participate in substrate binding and catalysis “Reusable” AA are not Sensitive to pH changes Denatured by high heat Inhibited by poison From: Harvey. Biochemistry Enzymes Part 1 PROPERTIES and CHARACTERISTICS OF ENZYMES Catalytic Efficiency: reactions Presence of Cofactor and coenzymes catalyzed by enzymes are 103-108 times faster than uncatalyzed reactions. Specificity: enzymes interact with one or very few substrates and catalyze only one type of chemical reaction. *Coenzymes and Cosubstrates are often the metabolically active form of the vitamins. PROPERTIES and CHARACTERISTICS OF ENZYMES Location in the cell: Many enzymes are in specific organelles in the cell (compartmentalization) and in specific cells some reactions are isolated from others (avoiding competition for the substrate or enabling more favorable conditions, like pH) Recall: protein sorting importance to maintain this compartmentalization. o Glycolysis o PP pathway o Fatty acid synthesis SERUM BIOQUEMISTRY - Enzymatic diagnosis Testing for specific enzymes provides information about the organs and tissues in the body as well as the metabolic state of the animal. Enzymes P1 FYI Liver Enzymes ALT alanine aminotransferase (typically found when the cells of the liver are stressed or damaged) ALP alkaline phosphatase (increased when bile flow in the liver is reduced) Pancreatic Enzymes Amylase Lipase PLI (pancreatic lipase immunoreactivity) Muscle Enzymes CK (creatine kinase) enzyme most frequently measured to assess injury (trauma, inflammation). AST (aspartate aminotransferase) and ALT (alanine aminotransferase) also used to assess liver function, lesser importance during muscle injury. PROPERTIES and CHARACTERISTICS OF ENZYMES Regulation: enzyme activity can be regulated (it can be increased or decreased) so that the rate of product (biochemical reactions) responds to cellular needs recall: insulin and glucagon regulating several enzymatic activities on metabolic map pathways Picture: ResearchGate How enzymes work https://www.youtube.com/watch?v=yk14dOOvwMk&t=1s Enzymes by Amoeba sisters https://www.youtube.com/watch?v=qgVFkRn8f10 HAPPY STUDYING Clara Camargo, DVM [email protected] ©2021 Ross University School of Veterinary Medicine. All rights reserved. Cellular Biology & Homeostasis ENZYMES - Part 2 VP 2024 Clara Camargo, DVM LEARNING OBJECTIVES Define enzyme kinetics and describe its relevance Explain Michaelis-Menten Kinetics (Vmax, Km, conclusions and curve) Understand the purpose of the Lineweaver-Burk plot, interpret 1/Vmax and 1/Km graphs Explain the concepts of enzyme inhibition: irreversible vs reversible, competitive and noncompetitive Explain competitive and non-competitive inhibitors using the Michaelis-Menten enzyme reaction curve and the Lineweaver-Burk plot List some examples of enzyme inhibitors ENZYME KINETICS The study of the chemical reactions that are catalyzed by enzymes. Studying enzyme kinetics allows for the understanding of how each enzyme works. o The enzyme’s catalytic mechanism o The control of enzyme activity o Its role in metabolism o Possible inhibition by drugs, agonists or antagonists Enzymes Part 2 Enzymes Part 2 MICHAELIS-MENTEN KINECTS Equation describes how reaction velocity varies with substrate concentration single substrate enzyme kinetics! An enzyme reversibly combines with its substrate to form an ES complex that yields a product P, releasing the free enzyme. Leonor Michaelis (1875-1949) Maud Menten (1879-1960) From: carnotcycle WordPress.com Assumptions/conditions: Michaelis-Menten kinetics Relative concentration of enzyme and substrate [S] > > % of total substrate bound by the enzyme at any one time is small Steady-state: ES complex does not change with time. Rate [ES] formation = [ES] disassociation Initial velocity (V0): reaction is measured as soon as enzyme and substrate are mixed. Concentration of P is very small. Rate of P ES can be ignored Total enzyme concentration [E] does not change over time. Michaelis Menten kinetics Km Michaelis-Menten constant Is characteristic of an enzyme and its particular substrate and reflects the affinity of the enzyme for that substrate. Km is equal to the substrate concentration at which the reaction velocity is ½ Vmax Km does not vary with enzyme concentration From: Harvey. Biochemistry CONCLUSIONS FROM MICHAELIS-MENTEN EQUATION: Michaelis Menten kinetics Enzyme 1 has a high affinity for its substrate because a low concentration of substrate is needed to half-saturate the enzyme this is represented in the small Km Enzyme 2 shows a low affinity to the substrate represented in the large Km CONCLUSIONS FROM MICHAELIS-MENTEN EQUATION: Michaelis Menten kinetics Relationship of velocity (V) to enzyme concentration: rate of the reaction is directly proportional to the enzyme concentration at all substrate concentrations. (if ½ [E] Order of reaction: First order [S] < Km the velocity of the reaction is nearly proportional to the substrate concentration. Zero order [S] > Km the velocity of the reaction is constant and equal to Vmax rate of reaction is independent of substrate concentration Michaelis Menten kinetics Michaelis Menten kinetics show hyperbolic curve (i.e.; myoglobin binding to O2 “single substrate”) 7 Menten kinetics; they show a sigmoidal curve (i.e.; hemoglobin binding to O2) O2 sat % enzyme w P O2 From: Harvey. Biochemistry Allosteric enzymes do not show Michaelis- more than one bindingsite Supplemental videos Role of Myoglobin https://www.youtube.com/watch?v=B8i9-Qh8q7Q&t=46s Myoglobin’s high O2 affinity https://www.youtube.com/watch?v=Y3GaZdrt8LQ Enzymes Part 2 THE LINEWEAVER-BURK PLOT reciprical of Michaelis constant When V0 is plotted against [S], it is not Mathematically, the reciprocal 1/v0 always possible to determine when Vmax is and 1/[S] will be plotted to obtain a achieved because of the hyperbolic curve. straight line Calculation of Km and Vmax enzyme activity It can also help to determine the mechanism of action of enzyme inhibitors Hans Lineweaver (1907-2009) and Dean Burk (1904-1988) From: Harvey. Biochemistry Enzymes Part 2 FACTORS AFFECTING REACTION VELOCITY noting In vitro studies information on how Substrate concentration: enzymes function in living cells living The rate of an enzyme catalyzed reaction Different enzymes show different increases with the substrate responses to changes in concentration until a maximal velocity substrate concentration Vmax ( mol/min) 2 addsubstrate to temperature van pH When (Vmax) is reached saturation (substrate bound to all available binding sites of enzymes) From: Harvey. Biochemistry Factors affecting reaction velocity Temperature: Reaction velocity increases with temperature until a peak is reached as TT Increase is the result of increased number of molecules having sufficient energy to pass over the energy activation barrier N Further elevation of the temperature results in a decrease in reaction velocity due to the denaturation of proteins The optimal temperature for most mammalian enzymes is 35-40°C (95-104 °F) Factors affecting reaction velocity pH: Extreme pH (concentration of H+) conditions can affect the reaction velocity The pH can affect the ionization state of the active site Extreme pH conditions can also denature enzymes (the structure of the catalytically active enzyme depends on the ionic character of the amino acid side chains) pH optimum of enzymes may vary (pepsin, trypsin, alkaline phosphatase) stomach smalstine INHIBITION OF ENZYME ACTIVITY Enzymes Part 2 block or INHIBITOR: any substance that can diminish the velocity of an enzyme-catalyzed reaction Irreversible (covalent bonds; inactivate enzymes, e.g. lead ferrochelatase) importantinthebuildupofher Reversible inhibitors bind to the enzyme through non-covalent bonds The dilution of the enzyme-inhibitor complex leads to the separation of the reversibly bound inhibitor, enzyme activity can be restored Competitive inhibition Noncompetitive inhibition Enzyme function and inhibition https://www.youtube.com/watch?v=PILzvT3spCQ COMPETITIVE INHIBITOR Inhibition of enzyme activity Binds reversibly to the same site that the substrate would normally bind, competing with substrate for that site. Competitive Inhibitors 1. Effect on Km: competitive inhibitor Km for given substrate competitive inhibitor reduces affinity of E for S (competing!!) More substrate is needed to reach ½ Vmax add more substrate reduced be of competitive inhibitors affinity solvesaffinity problem velocity remains the same 2. Effect on the Lineweaver-Burk plot 1. 1/Vmax = unchanged 2. 1/Km is higher in the presence of the competitive inhibitor 3. Effect on Vmax: inhibitor effect is reversed by increasing [S] High enough [S] reaction velocity reaches the same Vmax observed in the absence of inhibitor Enzymes Part 2 NONCOMPETITIVE INHIBITOR binds reversibly Binds at a site different from the substrate (allosteric site) It can bind the free enzyme or the ES complex preventing the reaction from occurring Of From: Harvey. Biochemistry Causes conformational change in enzyme or active site of the enzyme affinity inhibition to substrate stays the same Noncompetitive inhibition NONCOMPETITIVE INHIBITION From: Harvey. Biochemistry Max V will never be reached Decrease the Vmax of the reaction ( [S] does not reverse it – why?) Km remains unchanged as noncompetitive inhibitors do not interfere with the binding of substrate to the enzyme These effects can be readily seen when plotting these in a Lineweaver-Burk plot Enzymes Part 2 FYIfor VP dexdorm ketamine Enzymes Part 2 ENZYME INHIBITORS AS DRUGS At least half of ten most prescribed drugs in the U.S. are enzyme inhibitors -lactam antibiotics, such as penicillin and amoxicillin: act by inhibiting enzymes that are important for bacterial cell wall synthesis Angiotensin-converting enzyme (ACE) competitive inhibitors: block the enzyme that cleaves angiotensin I to the potent vasoconstrictor angiotensin II cause vasodilation lower blood pressure Aspirin: irreversibly inhibits prostaglandin and thromboxane synthesis STATIN DRUGS AS EXAMPLES OF COMPETITIVE INHIBITORS Competitive Inhibition Statins (atorvastatin, mevastatin and pravastatin) are structural analogs to this enzyme’s natural substrate Cholesterol biosynthesis Catalyzed by hydroxymethylglutarylCoA reductase (HMG-CoA reductase) prevent de novo cholesterol synthesis help lower plasma cholesterol levels From: Harvey. Biochemistry Veterinary Physiological Chemistry. Chapter 6: Enzymes kinetics HAPPY STUDYING Clara Camargo, DVM [email protected] ©2021 Ross University School of Veterinary Medicine. All rights reserved. Cellular Biology & Homeostasis ENZYMES - Part 3 VP 2024 Clara Camargo, DVM LEARNING OBJECTIVES 1. Explain cofactors (coenzyme, cosubstrate, prosthetic group) and give some examples 2. Understand what is a zymogen and its physiological relevance 3. Define apoptosis and generally explain how it works 4. Describe the mechanisms of enzyme regulation Enzymes Part 3 COFACTORS The catalytic activity of many enzymes depends on the presence of small Cofactor Enzyme Organic molecules Thiamine pyrophosphate Pyruvate dehydrogenase Flavin adenine dinucleotide (FADH) Monoamine oxidase molecules: cofactors Nicotinamide adenine dinucleotide (NADH) Lactate dehydrogenase small non-protein molecules Pyridoxal phosphate Glycogen phosphorylase Coenzyme A (CoA) Acetyl CoA carboxylase Biotin Pyruvate carboxylase “helpers” -Deoxyadenosyl cobalamin Tetrahydrofolate (THF) Methylmalonyl mutase Thymidylate synthase Metal Cofactors can be subdivided into two Zn 2+ Carbonic anhydrase Zn 2+ Carboxypeptidase 2+ EcoRV 2+ Hexokinase Mg groups: Mg Ni Urease 1. Inorganic metals Mo Nitrate reductase 2. Small organic molecules (coenzymes) 2+ Se Mn K + Glutathione peroxidase 2+ Superoxide dismutase Propionyl CoA carboxylase COFACTORS Enzymes Part 3 Cofactors Enzymes can exist in inactive forms (apoenzyme) and later be converted to active forms (holoenzyme) If the cofactor is a small organic molecule Apoenzyme Coenzyme binding Tightly bound (covalent bonds) with the help of a cofactor. coenzyme Holoenzyme prosthetic groups (Heme group is an example) Loosely bond co-substrates bind to and are released from the enzyme just as substrates and products are. Many enzymes acquire full enzymatic activity after they acquire the right folding (recall the posttranslational modifications). COENZYMES Enzymes Part 3 Coenzymes are often derived from vitamins. Can be either tightly or loosely bound to the enzyme. Are associated with the enzyme’s active site that assists with their catalytic function. Vitamins cannot be synthesized by humans and most animals; and must be supplied from the diet. Many vitamins are essential components of enzymes or provide those as coenzymes FYI COENZYMES - Biotin Enzymes Part 3 Attached to distinct lysine residues in Recall histones, affecting chromatin structure and mediating gene regulation (epigenetic modification) coenzymeformore than7 carboxylases ZYMOGENS Some enzymes are synthesized as inactive precursors that are activated by proteolytic cleavage of one or a few specific peptide bonds. The inactive precursor is called a zymogen (or a proenzyme). The biochemical change usually occurs in the Golgi apparatus, or when digestive enzymes are secreted in the organ lumen Enzymes Part 3 ENZYME ACTIVATION BY PROTEOLYSIS watch later Zymogens Specific proteolysis is common in cellular physiology The digestive enzymes that hydrolyze Many protein hormones are also proteins (proteases) are synthesized as synthesized as inactive precursors. zymogens in the stomach and pancreas. o o o (stomach) (proteolytic removal of a peptide) (pancreas) The pancreas secretes zymogens partly to Accidental activation of zymogens can prevent the enzymes from digesting happen when the secretion duct in the proteins in the cells in which they are pancreas is blocked by a gallstone synthesized. resulting in acute pancreatitis. SECRETION OF ZYMOGEN GRANULES BY CELLS OF THE PANCREAS Secretory function, these cells have many small granules of zymogens that are visible Darker-staining cells form clusters called acini, which are arranged in lobes separated by a thin fibrous barrier The secretory cells of each acinus surround a small intercalated duct EEE Zymogens ZYMOGENS Enzymes Part 3 Proteolytic cleavage = activation For the cleavage, energy (ATP) is not Proteolytic cleavage occurs just once in needed. the life of an enzyme molecule, the process is irreversible. Proteins (enzymes) located outside cells can be activated by proteolytic cleavage. Unlike allosteric control and reversible covalent modification DIETARY PROTEIN DIGESTION BY PROTEASES - Stomach Zymogens - Proteases HClacid to pepsins. Pepsinogens are converted in the gastric lumen by gastric o Once this reaction begins, pepsins can autocatalyze the conversion of pepsinogens to pepsins. 2 steps to regulate enzyme activation Gastric gland Zymogens The digestive enzymes that hydrolyze proteins are synthesized as zymOGEN or PROenzyme in the stomach and pancreas Cascade of events regulating digestive enzymes Stomach Proenteropeptidase Enteropeptidase Trypsinogen Chymotrypsinogen Proelastase Procarboxypeptidases A and B Pancreatic prolipase Site of synthesis Proenzyme/ Zymogen Active enzyme Stomach Pepsinogen Pepsin Pancreas Chymotrypsinogen Chymotrypsin Pancreas Trypsinogen Trypsin Pancreas Procarboxypeptidase Carboxypeptidase Pancreas Proelastase Elastase Proteins Pepsin Trypsinfirst afireenzyme Large peptides Gastric and pancreatic Chymotrypsin Elastase zymogens Carboxypeptidases A and B Pancreatic lipase Small peptides Aminopeptidases Dipeptidases Tripeptidases Free amino acids + Triglycerides ZYMOGEN AND COENZYME - COAGULATION CASCADE Blood clotting is mediated by a cascade of FYI proteolytic activations that ensures a rapid and amplified response to trauma. VITAMIN K (Coenzyme) FYI WARFARIN (epoxy reductase complex) ZYMOGEN - APOPTOSIS Programmed cell death, or apoptosis, is mediated by proteolytic enzymes called caspases, which are synthesized as zymogens (precursor form as procaspases) necrosis is non programmed cell death Unlike necrosis (traumatic cell death), apoptosis is highly regulated and does not cause inflammation When activated, caspases function to cause cell death in most multicellular organisms. It produces special cell fragments (apoptotic bodies), which are cleared by macrophages. Zymogens ZYMOGEN - APOPTOSIS Initiator caspases Zymogens executioner caspases kill the cell by degrading proteins indiscriminately Cannot stop once it has begun Can be initiated through: intrinsic pathway extrinsic pathway Both pathways use caspases (proteases) FYI OTHER ROLES OF ZYMOGENS IN BIOLOGY Zymogens Many developmental processes are controlled by the activation of zymogens. For example, in the metamorphosis of a tadpole into a frog, large amounts of collagen are resorbed from the tail. The conversion of procollagenase into collagenase (the active protease) is precisely timed in these remodeling processes. Likewise, much collagen is broken down in a mammalian uterus after delivery. From: Alberts. Mol. Biol. of the Cell ENZYME REGULATION MECHANISMS FOR REGULATING ENZYME ACTIVITY An organism must coordinate its different metabolic processes by: Regulating the reaction velocity of enzymes depending on the substrate concentration (Km range) (increased substrate Some enzymes with specialized regulatory functions can be regulated when physiologic conditions change, by changing: Enzyme activity Gene expression (Induction and repression of enzyme synthesis/degradation) Enzymes Part 3 MECHANISMS FOR REGULATING ENZYME ACTIVITY Enzymes Part 3 Cofactors/cosubstrates (can be reversible) Zymogens/proenzymes (irreversible activation) Enzyme activation cascades (irreversible once started) REGULATION OF ENZYME ACTIVITY Enzymes Regulation ALLOSTERIC ENZYMES Allosteric enzymes change shape upon Increase or decrease affinity for substrate binding of an effector. Effectors (modifiers/regulators) bind noncovalently at a site other than the active site. Altering the affinity of the enzyme for its substrate OR Modifying the maximal catalytic activity of the enzyme https://www.youtube.com/watch?v=ApKM-IkSElY REGULATION OF ENZYME ACTIVITY ALLOSTERIC ENZYMES Positive effector increase enzymatic activity Negative effector decreaseenzymatic activity Modify maximal catalytic velocity (Vmax) Effectors can influence affinity of enzyme for its substrate (K0.5) Both Enzymes Regulation Enzymes Regulation REGULATION OF ENZYME ACTIVITY ALLOSTERIC ENZYMES Homotropic effectors: when the substrate itself serves as an effector Most allosteric substrates function as positive homotropic effectors: the presence of the substrate molecule at one site of the enzyme enhances the catalytic properties of the other substrate-binding sites. Hemoglobin is a homotropic allosteric protein From: Harvey. Biochemistry REGULATION OF ENZYME ACTIVITY Enzymes Regulation ALLOSTERIC ENZYMES Heterotropic effectors: effector different from the substrate Classical example is a feedback inhibition of a metabolic pathway (end-product inhibition) Enzymes Regulation REGULATION OF ENZYME ACTIVITY COVALENT MODIFICATION (Reversible) Covalent modifications: usually addition or removal of phosphate groups from specific amino acids of the enzyme (Ser, Tyr, Thr). group phosphate 88k Phosphorylation reactions are catalyzed by kinases using ATP as a phosphate donor. The phosphorylated protein may be more or less active. i.e., glycogen metabolism REGULATION OF ENZYME ACTIVITY Enzymes Regulation ENZYME SYNTHESIS Cells can alter the rates of enzymes degradation or synthesis. Enzymes subject to synthesis regulation are often those that are needed at only one stage of life development or under certain physiological conditions. Induction or repression of protein synthesis are slow (hours to days), compared with allosteric or covalent regulation of enzyme activity. REGULATION OF ENZYME ACTIVITY - Summary Regulation of allosteric enzymes Covalent modification of enzymes Induction or repression of enzymes synthesis Enzymes Regulation HAPPY STUDYING Clara Camargo, DVM [email protected] ©2021 Ross University School of Veterinary Medicine. All rights reserved.