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عید سعید قربان بر شما مبارک یلدای زیبا هم همینطور! In the Name of Allah TUMS ENZYMES: …Make Life on Earth Possible Abolfazl Golestani, PhD An Important Question: • Why should we as medical students, study and learn about the ENZYMS? • For answer go to slide No. 55 5/23/2017 Enzymes; by: Dr. Abolfazl Golestani, PhD 3 Chemical reaction A Catalyst Product(s) Reactant(s) A +B B Catalyst B+C Catalysts •Increase the rate of a reaction •Are not consumed in the reaction •Can act repeatedly What are some of the known catalysts? 5/23/2017 Enzymes; by: Dr. Abolfazl Golestani, PhD Heat Acid Base Metals 4 Enzyme is either a pure protein or may require a non-protein portion Apoenzyme = protein portion Apoenzyme + non-protein part = Holoenzyme According to Holum, the non-protein portion may be: A coenzyme - a non-protein organic substance which is loosely attached to the protein part A prosthetic group - an organic substance which is firmly attached to the protein or apoenzyme portion A cofactor - these include K+, Fe++, Fe+++, Cu++, Co++, Zn++, Mn++, Mg++, Ca++, and Mo+++ 5/23/2017 Enzymes; by: Dr. Abolfazl Golestani, PhD 5 Basic enzyme reactions S+EE+P S = Substrate P = Product E = Enzyme Swedish chemist Savante Arrhenius in 1888 proposed: Substrate and enzyme form some intermediate known as the EnzymeSubstrate Complex (ES): S + E ES Binding step ES P + E Catalytic step 5/23/2017 Enzymes; by: Dr. Abolfazl Golestani, PhD 6 There are two models of enzyme substrate interaction 1. Lock and key model; Emil Fischer (1890) 2. Induced fit model; Daniel Koshland (1958) 5/23/2017 The active site: • Substrate Binding Site • Catalytic Site Enzymes; by: Dr. Abolfazl Golestani, PhD 7 Induced fit in Carboxypeptidase A Three amino acids are located near the active site (Arg 145, Tyr 248, and Glu 270) 5/23/2017 Enzymes; by: Dr. Abolfazl Golestani, PhD 8 Enzyme-Substrate complex is transient S+E S E P+E When the enzyme unites with the substrate, in most cases the forces that hold the enzyme and substrate are noncovalent. Binding forces of substrate are: Ionic interactions: (+)•••••(-) Hydrophobic interactions: (h)•••••(h) H-bonds: O-H ••••• O, N-H ••••• O, etc. van der Waals interactions 5/23/2017 Enzymes; by: Dr. Abolfazl Golestani, PhD 9 Some important characteristics of enzymes -Potent (high catalytic power) High reaction rates They increase the rate of reaction by a factor of 103-1012 -Efficient (high efficiency) catalytic efficiency is represented by Turnover number: moles of substrate converted to product per second per mole of the active site of the enzyme -Milder reaction conditions Enzymatically catalyzed reactions occur at mild temperature, pressure, and nearly neutral pH (i.e. physiological conditions) 5/23/2017 Enzymes; by: Dr. Abolfazl Golestani, PhD 10 Some important characteristics of enzymes, cont. -Specific (specificity) Substrate specific Reaction Specific Stereospecific -Capacity for regulation Enzymes can be activated or inhibited so that the rate of product formation responds to the needs of the cell -Location within the cell Many enzymes are located in specific organelles within the cell. Such compartmentalization serves: to isolate the reaction substrate from competing reactions, to provide a favorable environment for the reaction, and to organize the thousands of enzymes present in the cell into purposeful pathways. 5/23/2017 Enzymes; by: Dr. Abolfazl Golestani, PhD 11 Specificity Substrate Specificity Absolute specificity: For example Urease Functional Group specificity: For example OH, CHO, NH2. Linkage specificity: For example Peptide bond. Reaction specificity Yields are nearly 100% Lack of production of by-products Save energy and prevents waste of metabolites Stereospecificity Enzymes can distinguish between enantiomers and isomers 5/23/2017 Enzymes; by: Dr. Abolfazl Golestani, PhD 12 Enzymes requiring metal ions as cofactors 5/23/2017 Enzymes; by: Dr. Abolfazl Golestani, PhD 13 Many Vitamins vitamins are are Coenzyme coenzyme precursors Many precursors • Many organism are unable to synthesize parts of the coenzymes • These parts must be present in the organism diet and are called vitamins Vitamin Chemical Name Biochemical Function Coenzyme Chemistry B1 Thiamine Coenzyme TPP Decarboxylation of - keto acids B2 Riboflavin Coenzymes FAD, FMN Redox chemistry Niacin Nicotinamide Coenzyme NAD Redox chemistry B6 Pyridoxal Coenzyme PLP Transamination reactions B12 Cobalamine Coenzyme B12 Radical rearrangements (lipid degradation) C Ascorbic Acid Coenzyme Redox agent ( collagen formation) H 5/23/2017 Biotin Enzymes;Coenzyme by: Dr. Abolfazl Golestani, PhD Carboxylation 14 Methods for naming enzymes (nomenclature) • • • Very old method: Pepsin, Renin, Trypsin Old method: Protease, Lipase, Urease Systematic naming (EC = Enzyme Commission number): • • • The name has two parts: The first part: name of substrate (s) The second part: ending in –ase, indicates the type of reaction. Additional information can follow in parentheses: • L-malate:NAD+ oxidoreductase (decarboxylating) 5/23/2017 Enzymes; by: Dr. Abolfazl Golestani, PhD 15 Each enzyme has an EC number = Enzyme Commission number Enzyme EC number Alcohol dehydrogenase Arginase 1.1.1.1 3.5.3.1 Pepsin 3.4.21.1 • EC number consists of 4 integers: • The 1st designates to which of the six major classes an enzyme belongs • The 2nd integer indicates a sub class, e.g. type of bond • The 3rd number is a subclassification of the bond type or the group transferred in the reaction or both (a subsubclass) • The 4th number is simply a serial number 5/23/2017 Enzymes; by: Dr. Abolfazl Golestani, PhD 16 There are six functional classes of enzymes Class Names Functions 1 Oxidoreductases AH + NAD+ A+ + NADH 2 Transferases A-X + B A + B-X 3 Hydrolases A-OX + H2O A-OH + HOX 4 Lyases R1R2R3CCR4R5R6 R1R2C=CR4R5 + R3 + R6 5 Isomerases trans cis, L-form D-form, etc. 6 Ligases Formation of C-C, C-S, C-O, C-N bonds by condensation reactions coupled to ATP hydrolysis 5/23/2017 Enzymes; by: Dr. Abolfazl Golestani, PhD 17 EC3 Hydrolases Function EC 3.1 Acting on ester bonds EC 3.2 Glycosylases EC 3.3 Acting on ether bonds EC 3.4 EC 3.5 EC5 Isomerases Function EC 5.1 Racemases and epimerases Acting on peptide bonds (peptidases) EC 5.2 cis-transIsomerases Acting on carbon-nitrogen bonds, other than peptide bonds EC 5.3 Intramolecular isomerases EC 5.4 Intramolecular transferases (mutases) EC 5.5 Intramolecular lyases EC 5.99 Other isomerases EC 3.6 Acting on acid anhydrides EC 3.7 Acting on carbon-carbon bonds EC 3.8 Acting on halide bonds EC 3.9 Acting on phosphorusnitrogen bonds EC 3.10 Acting on sulfur-nitrogen bonds EC5/23/2017 Acting on carbon-phosphorus Enzymes; by: Dr. Abolfazl Golestani, PhD 3.11 bonds 18 Enzyme Nomenclature and Classification EC Classification Class Subclass Sub-subclass Serial number 5/23/2017 Enzymes; by: Dr. Abolfazl Golestani, PhD 19 Example of Enzyme Nomenclature • Common name(s): Invertase, sucrase • Systematic name: -D-fructofuranoside fructohydrolase (E.C. 3.2.1.26) • Recommended name: 5/23/2017 Enzymes; by: Dr. Abolfazl -fructofuranosidase Golestani, PhD 20 Kinetic 5/23/2017 Enzymes; by: Dr. Abolfazl Golestani, PhD 21 Energy barrier = Free Energy of Activation X T* Y T = Transition state (Ea) Thermodynamics: Type (Exergonic or Endergonic) Kinetics: How fast the reaction will proceed 5/23/2017 Enzymes; by: Dr. Abolfazl Golestani, PhD 22 Enzyme Stabilizes Transition State What’s the difference? Many enzymes function Enzymes; by: Dr. Abolfazl by5/23/2017 lowering the activation energy of reactions. Golestani, PhD 23 Adapted from Alberts et al (2002) Molecular Biology of the Cell (4e) p.166 عید سعید و عظیم غدیربرشما مبارک ميالد حضرت امام هادي (ع) را تهنيت مي گويم، میالد حضرت مسیح (ع) را هم همین طور! EA = Activation energy; a barrier to the reaction Can be overcome by adding energy....... ......or by catalysis 5/23/2017 Enzymes; by: Dr. Abolfazl Golestani, PhD 25 Enzymes Are Complementary to Transition State X If enzyme just binds substrate then there will be no further reaction Enzyme not only recognizes substrate, 5/23/2017 Enzymes; Dr. Abolfazlstate, see also Enz01 but also induces the formation ofby: transition Golestani, PhD 26 Active Site Is a Deep Buried Pocket Why energy required to reach transition state is lower in the active site? It is a magic pocket + CoE (1) (4) (3) 5/23/2017 (2) (1) Stabilizes transition (2) Expels water (3) Reactive groups (4) Coenzyme helps Enzymes; by: Dr. Abolfazl Golestani, PhD 27 Juang RH (2004) BCbasics Active Site Avoids the Influence of Water + - Preventing the influence of water sustains the formation of stable ionic bonds 5/23/2017 Enzymes; by: Dr. Abolfazl Golestani, PhD 28 Adapted from Alberts et al (2002) Molecular Biology of the Cell (4e) p.115 Enzyme Reaction Mechanism o Consider for example the mechanism of Chymotrypsin: o Enz06 o Enz07 5/23/2017 Enzymes; by: Dr. Abolfazl Golestani, PhD 29 Modes of rate enhancement Facilitation of Proximity Increase the Effective concentration Hold reactants near each other in proper orientation Strain, Molecular Distortion, and Shape Change Put a strain on susceptible bonds General Acid –Base Catalysis Transfer of a proton in the transition state Covalent Catalysis Form covalent bond with substrate destabilization of the substrate 5/23/2017 Enzymes; by: Dr. Abolfazl Golestani, PhD 30 Factors Affecting Rate of Enzyme Reactions Temperature pH Enzyme concentration [E] Substrate concentration [S] Inhibition Regulation (Effectors) 5/23/2017 Enzymes; by: Dr. Abolfazl Golestani, PhD 31 1- Optimum Temperature Little activity at low temperature (low number of collisions) Rate increases with temperature (more successful collisions); rate doubles for every 10°C increase in temperature Most active at optimum temperatures (usually 37 oC in humans) Enzymes isolated from thermophilic organisms display maxima around 100 °C Enzymes isolated from psychrophilic organisms display maxima around 10 °C. Activity lost with denaturation at high temperatures 5/23/2017 Enzymes; by: Dr. Abolfazl Golestani, PhD 32 2- Optimum pH • Effect of pH on ionization of active site • Effect of pH on enzyme denaturation • Each enzyme has an optimal pH (~ 6 - 8 ) – Exceptions : digestive enzymes in the stomach (pH 2) digestive enzymes in the intestine (pH 8) 5/23/2017 Enzymes; by: Dr. Abolfazl Golestani, PhD 33 3- Enzyme concentration • The Rate (v) of reaction Increases proportional to the enzyme concentration [E] ([S] is high) 5/23/2017 Enzymes; by: Dr. Abolfazl Golestani, PhD 34 4- Substrate concentration • When enzyme concentration is constant, increasing [S] increases the rate of reaction, BUT • Maximum activity reaches when all E combines with S (when all the enzyme is in the ES form) 5/23/2017 Enzymes; by: Dr. Abolfazl Golestani, PhD 35 Enzyme Velocity Curve, see also Enz02 0 1 2 3 4 5 6 7 8 S + E 80 60 Product (v) 40 (in a fixed period of time) 20 0 5/23/2017 P 0 2 4 6 Substrate (mole) [S] Enzymes; by: Dr. Abolfazl Golestani, PhD 8 36 Juang RH (2004) BCbasics بسم هللا الرحمن الرحیم Enzymes rd 3 part 5/23/2017 Enzymes; by: Dr. Abolfazl Golestani, PhD 37 Michaelis-Menten Equation S K+1 E k-1 S E k2 P maximal velocity, Vmax 5 v, µmol/min 4 3 0.5Vmax 2 Km 1 0 0 5/23/2017 10 20 30 [S], mM 40 50 Enzymes; by: Dr. Abolfazl Golestani, PhD 38 MM Equation Derivation (steady state) 5/23/2017 Enzymes; by: Dr. Abolfazl Golestani, PhD 39 Practical Summary - Vmax and Km • Vmax – How fast the reaction can occur under ideal circumstances • Km – Range of [S] at which a reaction will occur – Binding affinity of enzyme for substrate • LARGER Km the WEAKER the binding affinity 5/23/2017 Enzymes; by: Dr. Abolfazl Golestani, PhD 40 Practical Summary, cont. Enzyme Substrate Km (mM) Catalase H2O2 1,100 Chymotrypsin Gly-Tyr-Gly 108 Carbonic anhydrase CO2 12 Beta-galactosidase D-lactose 4 Acetylcholinesterase acetylcholine (ACh) 0.09 5/23/2017 Enzymes; by: Dr. Abolfazl Golestani, PhD 41 Practical Summary; cont. • Kcat/Km – Practical idea of the catalytic efficiency, i.e. how often a molecule of substrate that is bound reacts to give product 5/23/2017 Enzymes; by: Dr. Abolfazl Golestani, PhD 42 Order of Reaction 1. When [S] << Km vo = (Vmax/Km )[S] 2. When [S] = Km vo = Vmax/2 3. When [S] >> Km vo = Vmax 5/23/2017 zero order Mixed order 2 First order Enzymes; by: Dr. Abolfazl Golestani, PhD 43 Importance of Vi in Measurement of Enzyme Activity S E k1 k-1 k2 S E P Working with vo minimizes complications with 1. reverse reactions 2. product Inhibition The rate of the reaction catalyzed by an enzyme in a sample is expressed in Units. Units = V = activity = Micromoles (mol; 10-6 mol or ….), of substrate reacting or product produced per min. It is better to measure it at linear part of the curve 5/23/2017 Enzymes; by: Dr. Abolfazl Golestani, PhD 44 Lineweaver-Burk plot 1 vo -1 Km vo 1/2 1 Vmax 1 Km 1 1 v Vmax [S] Vmax 5/23/2017 Km Direct plot S 1/S Vmax [S] v Km [S] Enzymes; by: Dr. Abolfazl Golestani, PhD 45 Juang RH (2004) BCbasics Double reciprocal plot Allosteric Enzymes • Why the sigmoid shape? • Allosteric enzymes are multi-subunit enzymes, each with an active site • They show a cooperative response to substrates • See Enz13 hyperbolic curve; Michaelis-Menten kinetics Sigmoidal curve 5/23/2017 Enzymes; by: Dr. Abolfazl Golestani, PhD 46 Irreversible Inhibition=Enzyme Stops Working Permanently 1. 2. Destruction of enzyme Irreversible Inhibitor=forms covalent bonds to E (inactive E) Examples: – Diisopropylfluorophosphate • inhibits acetylcholine esterase • binds irreversibly to –OH of serine residue – Cyanide and sulfide • Inhibit cytochrome oxidase • bind to the iron atom – Fluorouracil • inhibits thymidine synthase (suicide inhibition - metabolic product is toxic ) – Aspirin • Inhibits prostaglandin synthase • acylates an amino group of the cyclooxygenase 5/23/2017 Enzymes; by: Dr. Abolfazl Golestani, PhD 47 Reversible Inhibition=Temporary Decrease of Enzyme Function • Three types based on “how increasing [S] affects degree of inhibition”: 1. Competitive: degree of inhibition decreases 2. Non-competitive: degree of inhibition is unaffected 3. Anti- or Uncompetitive: degree of inhibition increases The Lineweaver-Burk plot is useful in determining the mechanisms of actions of various inhibitors, see Enz04 5/23/2017 Enzymes; by: Dr. Abolfazl Golestani, PhD 48 The Effects of Enzyme Inhibitors 5/23/2017 Enzymes; by: Dr. Abolfazl Golestani, PhD 49 Example • When a slice of apple is exposed to air, it quickly turns brown. This is because the enzyme o-diphenyl oxidase catalyzes the oxidation of phenols in the apple to dark-colored products. • Catechol can be used as the substrate. The enzyme converts it into o-quinone (A), which is then further oxidized to dark products. 5/23/2017 Enzymes; by: Dr. Abolfazl Golestani, PhD 50 Experiments No Inhibitor Tube A Tube B Tube C Tube D [S] 4.8 mM 1.2 mM 0.6 mM 0.3 mM 1/[S] 0.21 0.83 1.67 3.33 Δ OD540 (Vi) 0.081 0.048 0.035 0.020 1/Vi 12.3 Tube A [S] 4.8 mM 20.8 Tube B 1.2 mM 31.7 Tube C 0.6 mM Tube A Tube B Tube C Tube D [S] 4.8 mM 1.2 mM 0.6 mM 0.3 mM 1/[S] 0.21 0.83 1.67 3.33 ΔOD540 (Vi) 0.060 0.032 0.019 0.011 1/Vi 16.7 31.3 52.6 90.9 50.0 Tube D 0.3 mM 1/[S] 0.21 0.83 1.67 3.33 ΔOD540 (Vi) 0.040 0.024 0.016 0.010 1/Vi 25 41 62 102 5/23/2017 effect of para-hydroxybenzoic acid (PHBA) effect of phenylthiourea Enzymes; by: Dr. Abolfazl Golestani, PhD 51 5/23/2017 Enzymes; by: Dr. Abolfazl Golestani, PhD 52 I- Competitive Inhibition EI S Competitive V [S] v max Km [S] CI V [S] v max K m [S] Ki E Sc + E + I ES 1 Km 1 1 v Vmax [S] Vmax 5 E+P 1 K m 1 1 v Vmax [S] Vmax 2.5 No I 4 v, µmol/min µmol/min v, 0.5V max +CI 0.5V max 3 2 K m Kmapp 1 -1/Km app +CI 2 1/v, /µmol/min /µmol/min 1/v, [I] 1 Kic -1/Km -1/K m Km Kmapp/Vmax 1.5 1 0.5 0 K /V K m /V max m max No I 1/V 1/V max max 0 0 5/23/2017 10 20 30 [S], mM 40 50 -0.6 -0.4 -0.2 Enzymes; by: Dr. Abolfazl Golestani, PhD 0 0.2 0.4 0.6 0.8 1/[S], /mM 1 53 II- Noncompetitive Inhibition S Noncompetitive (mixed-type) NCI S V [S] v max Km [S] E Vmax [S] v K m ' [S] EI E Kic S+E + I NCI 1 Km 1 1 v Vmax [S] Vmax 55 E+P 1 K m 1 ' v Vmax [S] Vmax 2.5 44 0.5Vmax 33 + NCImax 0.5V 0.5Vmax 22 K 11 Km [I] 1 Kic 2 1/v, /µmol/min /µmol/min 1/v, No I v, µmol/min ESI Kiu ES + I [I] ' 1 Kiu -1/K m -1/K m m 1.5 1/Vmaxapp Km/Vmaxapp + NC I 1 0.5 K K/V /V m mmax max 1/V max 1/V 00 No I max 0 00 5/23/2017 10 10 20 20 30 30 [S], [S], mM mM 40 40 50 50 -0.6 -0.4 -0.2 Enzymes; by: Dr. Abolfazl Golestani, PhD 0 0.2 0.4 0.6 0.8 1/[S], /mM 1 54 III- Uncompetitive Inhibition Uncompetitive (catalytic) Vmax [S] v Km [S] S E Vmax [S] v Km ' [S] ESI Kiu UCI S+E ES E+P + I 1 Km 1 1 1 Km 1 ' v Vmax [S] Vmax v Vmax [S] Vmax 5 2.5 No I 4 2 max + UC I 0.5V max 3 Km 2 K 1 0.5Vmax [I] ' 1 Kiu app m -1/Km Km -1/Km app-1/Km 1/v, /µmol/min v, µmol/min 0.5V 1/Vmaxapp 1.5 Kmapp/Vmaxapp 1 0.5 1/Vmax K /V m max + UC I Km/Vmax No I 1/Vmax 0 0 0 5/23/2017 10 20 30 [S], mM [S]. 40 50 -0.6 -0.4 -0.2 Enzymes; by: Dr. Abolfazl Golestani, PhD 0 0.2 0.4 0.6 0.8 1/[S]. /mM 1/[S], 1 55 Enzyme Inhibitors in Medicine • Many current pharmaceuticals are enzyme inhibitors (e.g. HIV protease inhibitors for treatment of AIDS) • An example: Ethanol is used as a competitive inhibitor to treat methanol poisoning Methanol Alcohol dehydrogenase formaldehyde (very toxic) Ethanol competes for the same enzyme Administration of ethanol occupies the enzyme thereby delaying methanol metabolism long enough for clearance through the kidneys 5/23/2017 Enzymes; by: Dr. Abolfazl Golestani, PhD 56 Some diagnostically important enzymes Aminotransferases Aspartate aminotransferase (AST or SGOT) Alanine aminotransferase (ALT, or SGPT) Myocardial infarction Viral hepatitis Lactate Dehydrogenase (LDH) myocardial infarction Creatine Kinase (CK) Myocardial infarc., brain, skeletal muscle disorder Cholinesterase Liver, erythrocytes Gamma-glutamyltransferase Liver damage Acid phosphatase Carcinoma of prostate Alkaline phosphatase (AP) Bone disease Lipase Acute pancreatitis Ceruloplasmin Hepatolenticular degeneration (wilson’s disease) Alpha-amylase Intestinal obstruction 5/23/2017 Enzymes; by: Dr. Abolfazl Golestani, PhD 58 5/23/2017 Useful enzymes for early diagnosis of dental caries and periodontal disease Enzymes; by: Dr. Abolfazl Golestani, PhD 59 Isozymes of Lactate Dehydrogenase Isozymes: – Are catalitically identical (have same catalytic activity) BUT physically distinct – Can be detected by gel electrophoresis (different electrical charge) – Occur in oligomeric enzymes like lactate dehydrogenase (LDH) In LDH • Protomers H and M can combine to make five different tetramers. 5/23/2017 Enzymes; by: Dr. Abolfazl Golestani, PhD 60 Isoenzymes of Creatine Kinase • CK has 3 forms dimer B and M chains: • CK1= BB • CK2= MB • CK3=MM • Heart, the only tissue rich in CK2, increases 4-8 hr after chest pains- peaks at 24 hr. • LDH peaks 2-3 days after MI. • New markers: Troponin T, Troponin I 5/23/2017 Enzymes; by: Dr. Abolfazl Golestani, PhD 61 5/23/2017 Enzymes; by: Dr. Abolfazl Golestani, PhD 62 5- Regulation (Effectors) Effectors can be classified as follows: According to type: • Homotropic effector: Substrate itself is the effector • Heterotropic effector: substance other than substrate is the effector According to their effect: • Activators (positive effectors) – Increase the rate of enzyme • Inhibitors (negative effectors) – Decrease the velocity of reaction – Stop the enzyme Increase or decrease in enzyme reaction rate is reflected in the graph of V versus S • Irreversible • Reversible – Competitive – Non-competitive 5/23/2017– Uncompetitive Enzymes; by: Dr. Abolfazl Golestani, PhD 63 Metabolic Pathways • A metabolic pathway is a chain of enzymatic reactions – Most pathways have many steps, each having a different enzyme (E1, E2, E3, E4) – Step by step, the initial substance used as substrate by the first enzyme is transformed into a product that will be the substrate for the next reaction • Metabolic regulation is necessary to: – maintain cell components at appropriate levels. – conserve materials and energy. 5/23/2017 Enzymes; by: Dr. Abolfazl Golestani, PhD 64 Regulation of “Enzyme Activity” A. Regulation at transcription level (slowest) B. Isozymes: enzymes specific for distinct tissues and developmental stages C. Compartmentation of S, E and P D. Specific proteolytic cleavage E. Covalent modification (Reversible phosphorylation or adenylation) F. In response to metabolic products (fastest) 1. 2. 3. 4. Substrate level control Product Inhibition Feedback control Allosteric Effectors 5/23/2017 Enzymes; by: Dr. Abolfazl Golestani, PhD 65 A. Regulation at Transcription Level 1. Regulation of [E] by • Gene repression • Induction of genetic expression of enzyme 2. There is competition in a cell between the processes of protein synthesis and protein destruction • By altering these rates, one can alter the whole cell catalytic rate 3. It is rather slow 5/23/2017 Enzymes; by: Dr. Abolfazl Golestani, PhD 66 B. Isoenzymes • Isozymes provide a means of regulation, specific to distinct tissues and developmental stages • Differential expression of isozymes • LDH (for example) • Preferential substrate affinity 5/23/2017 Enzymes; by: Dr. Abolfazl Golestani, PhD 67 C. Compartmentalization of enzymes Substrates and cofactors within the cell are also compartmentalized Examples: • Enzymes of glycolysis are located in the cytoplasm • Enzymes of citric acid cycle are in the mitochondria • Hydrolytic enzymes are found in the lysosome 5/23/2017 Enzymes; by: Dr. Abolfazl Golestani, PhD 68 D. Proteolytic activation Activation of a zymogen • Some enzymes are secreted as inactive precursors, called zymogens. • Pancreatic proteases - trypsin, chymotrypsin, elastase, carboxypeptidase are all synthesized as zymogens: trypsinogen, chymotrypsinogen, proelastase and procarboypeptidase • Clotting factors are also part of a proteolytic cascade • Hormone peptides (Pro-insulin Insulin) • An on/off switch more than regulation 5/23/2017 Enzymes; by: Dr. Abolfazl Golestani, PhD 69 E. Covalent modification Reversible phosphorylation Phosphorylation is the most common type of modification. Two important classes of enzymes are: – Kinases Add a phosphate group to another protein/enzyme (phosphorylation) • transfer of phosphoryl group from ATP to -OH group of serine, threonine or tyrosine – Phosphatases Remove a phosphate group from a protein/enzyme (dephosphorylation) 5/23/2017 Enzymes; by: Dr. Abolfazl Golestani, PhD 70 1- Control of [S] • Concentration of substrate and product also control the rate of reaction, providing a biofeedback mechanism • Usually: 0.1 Km<[SPhysiologic]<10 km Mild changes in [S] Change in enzyme activity Homotropic effectors – substrate itself (binding at different site other than the active site) affects enzyme activity on other substrate molecules. Most often this is a positive effector. 5/23/2017 Enzymes; by: Dr. Abolfazl Golestani, PhD 71 2- Product inhibition • Enzyme is reversibly inhibited by the product Example: hexokinase in the first reaction of glycolysis is inhibited by glucose-6-phosphate (G6P; the product) glucose + ATP glucose-6-phosphate + ADP _ Why? As v approaches Vmax, the product becomes significant, and can compete with the substrate for the enzyme. The product becomes a competitive inhibitor and slows down activity of the enzyme. 5/23/2017 Enzymes; by: Dr. Abolfazl Golestani, PhD 72 3- Negative feedback control (end product inhibition) • Final product of a metabolic sequence feeds-back negatively on early steps • In feedback inhibition, there is a second binding site on the enzyme where the inhibitor binds, so that the inhibitor is not necessarily similar in structure to the substrate Enz 1 A _ Enz 2 B Enz 3 C Enz 4 D E What happens? • • • As the need for product E decreases, E will accumulate Most efficient to inhibit at first step of the pathway, slow the first reaction so intermediates do not build up An increase in the concentration of E, leads to a decrease in its rate of production of E 5/23/2017 Enzymes; by: Dr. Abolfazl Golestani, PhD 73 Regulation of the metabolism, feed-back inhibition by the final product - end product inhibition 1. Simple feed-back inhibition. The final product (E) inhibits the step from A to B. 2. Co-operative feed-back inhibition. Both final products (D, E) inhibit the first step of their own synthesis together. 3. Multivalent feed-back inhibition. 5/23/2017 4. Inhibition at a ramification of a biosynthesis pathway (sequential inhibition) 74 Enzymes; by: Dr. Abolfazl Golestani, PhD 4- Positive feedforward control • Earlier reactants in a metabolic sequence feed-forward positively on later steps. + If A is accumulating, it speeds up downstream reactions to use it up + Metabolism involves the complex integration of many feedback and feedforward loops 5/23/2017 Enzymes; by: Dr. Abolfazl Golestani, PhD 75 4- Allosteric control • Allosteric activator stabilizes active "R" state – shift the graph to the left • Allosteric inhibitor stabilizes less active or inactive "T" state – shift the graph to the right 5/23/2017 Enzymes; by: Dr. Abolfazl Golestani, PhD 76 5/23/2017 Enzymes; by: Dr. Abolfazl Golestani, PhD 77 Multi reactant enzymes reactancy • Published by W. W. Cleland in1963 • Nomenclature is based on number of substrates and products in the reaction. • Reactancy: the number of kinetically significant substrates or products and designated by syllables Uni, Bi, Ter, Quad. 5/23/2017 AP Uni Uni AP+Q Uni Bi A+BP+Q Bi Bi A+B+CP+Q+R+S Ter Quad Enzymes; by: Dr. Abolfazl Golestani, PhD 78 Multi reactant enzymes mechanism Sequential - if all S add to E before any P are released. – Sequential ordered - if S add in an obligatory order (two on; two off) – Sequential random - if S do not add in obligatory order (two on; two off) Ping Pong - If one or more S released before all S bind • (one on, one off; one on, one off); • Note: there is some sort of modified enzyme intermediate (often covalent intermediate) 5/23/2017 Enzymes; by: Dr. Abolfazl Golestani, PhD 79 5/23/2017 Enzymes; by: Dr. Abolfazl Golestani, PhD 80 Random sequential (example) 5/23/2017 Enzymes; by: Dr. Abolfazl Golestani, PhD 81 Ordered sequential (example) 5/23/2017 Enzymes; by: Dr. Abolfazl Golestani, PhD 82 Ping pong or double displacement mechanism 5/23/2017 Enzymes; by: Dr. Abolfazl Golestani, PhD 83 Double displacement (example) 5/23/2017 Enzymes; by: Dr. Abolfazl Golestani, PhD 84 Other kinds of enzymes • Some ribonucleoprotein enzymes have been discovered –The catalytic activity is in the RNA part –They are called Ribozymes • Catalytic antibodies are called Abzymes 5/23/2017 Enzymes; by: Dr. Abolfazl Golestani, PhD 85 موفق باشید 5/23/2017 Enzymes; by: Dr. Abolfazl Golestani, PhD 86