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TUMS Azin Nowrouzi, PhD Tehran University of Medical Sciences Chemical reaction A Catalyst Product(s) Reactant(s) A +B B Catalyst B+C Catalysts •Increase the rate of a reaction. •Are not consumed by the reaction. •Can act repeatedly. What are some of the known catalysts? Heat Acid Base Metals 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+++. 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 substance known as The Enzyme-Substrate Complex (ES): S + E ES Binding step ES P + E Catalytic step There are two models of enzyme substrate interaction 1. Lock and key model Emil Fischer (1890) 2. Induced fit model Daniel Koshland (1958) The active site: • Substrate Binding Site • Catalytic Site Induced fit in Carboxypeptidase A Three amino acids are located near the active site (Arg 145, Tyr 248, and Glu 270) 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 Some important characteristics of enzymes 1. Potent (high catalytic power) High reaction rates – 2. They increase the rate of reaction by a factor of 103-1012 Efficient (high efficiency) – catalytic efficiency is represented by Turnover number. • 3. 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) 4. Specific (specificity) – – – 5. 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. 6. Location within the cell Many enzymes are located in specific organelles within the cell. Such compartmentization 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 pathqways. Specificity • Substrate Specificity 1. Absolute specificity: For example Urease 2. Functional Group specificity: For example OH, CHO, NH2. 3. 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 Enzymes requiring metal ions as cofactors Many vitamins are coenzyme precursors Many Vitamins are Coenzyme 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 Biotin Coenzyme Carboxylation Methods for naming enzymes (nomenclature) 1. Very old method: Pepsin, Renin, Trypsin 2. Old method: Protease, Lipase, Urease 3. 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) Each enzyme has a 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 susubclass) • The 4th number is simply a serial number 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 reaction coupled to ATP hydrolysis EC 3 Hydrolases Function EC 3.1 Acting on ester bonds EC 3.2 Glycosylases EC 3.3 Acting on ether bonds EC 3.4 Acting on peptide bonds (peptidases) EC 3.5 Acting on carbon-nitrogen bonds, other than peptide bonds 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 phosphorus-nitrogen bonds EC 3.10 Acting on sulfur-nitrogen bonds EC 3.11 Acting on carbon-phosphorus bonds EC 3.12 Acting on sulfur-sulfur bonds EC 3.13 Acting on carbon-sulfur bonds EC5 Isomerases EC 5.1 Racemases and epimerases EC 5.2 cis-trans-Isomerases EC 5.3 Intramolecular isomerases EC 5.4 Intramolecular transferases (mutases) EC 5.5 Intramolecular lyases EC 5.99 Other isomerases Enzyme Nomenclature and Classification EC Classification Class Subclass Sub-subclass Serial number Example of Enzyme Nomenclature • Common name(s) – Invertase, sucrase • Systematic name – -D-fructofuranoside fructohydrolase (E.C. 3.2.1.26) • Recommended name – -fructofuranosidase Kinetic 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 Enzyme Stabilizes Transition State What’s the difference? Many enzymes function by lowering the activation energy of reactions. Adapted from Alberts et al (2002) Molecular Biology of the Cell (4e) p.166 EA = Activation energy ; a barrier to the reaction Can be overcome by adding energy....... ......or by catalysis Enzymes Are Complementary to Transition State X If enzyme just binds substrate then there will be no further reaction Enzyme not only recognizes substrate, but also induces the formation of transition state Active Site Is a Deep Buried Pocket Why energy required to reach transition state is lower in the active site? + CoE (1) (4) (3) (2) (1) Stabilizes transition (2) Expels water (3) Reactive groups (4) Coenzyme helps Juang RH (2004) BCbasics Active Site Avoids the Influence of Water + - Preventing the influence of water sustains the formation of stable ionic bonds Adapted from Alberts et al (2002) Molecular Biology of the Cell (4e) p.115 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 of the substrate. destabilization Factors affecting rate of enzyme reactions 1. 2. 3. 4. 5. 6. Temperature pH Enzyme concentration [E] Substrate concentration [S] Inhibition Regulation (Effectors) 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 C 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 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 intestine (pH 8) 3- Enzyme concentration • The Rate (v) of reaction Increases proportional to the enzyme concentration [E] ([S] is high). 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). Enzyme Velocity Curve 0 1 2 3 4 5 6 7 8 S + E 80 Product (v) 60 P 40 (in a fixed period of time) 20 0 0 2 4 6 8 Substrate (mole) [S] Juang RH (2004) BCbasics Michaelis-Menten equation S k1 E k-1 maximal velocity, Vmax 5 v, µmol/min 4 3 0.5Vmax 2 Km 1 0 0 10 20 30 [S], mM 40 50 S E k2 P MM equation derivation (steady state) 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 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 • Kcat / Km – Practical idea of the catalytic efficiency, i.e. how often a molecule of substrate that is bound reacts to give product. Order of reaction 1. When [S] << Km vo = (Vmax / Km )[S] 2. When [S] = Km vo = Vmax /2 3. When [S] >> Km vo = Vmax zero order Mixed order 2 First order 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 [S] reacting or [P] produced/min. It is better to measure it at linear part of the curve Lineweaver-Burk plot 1 vo -1 Km vo 1/2 1 Vmax 1 Km 1 1 v Vmax [S] Vmax Km Direct plot S Vmax [S] v Km [S] Juang RH (2004) BCbasics Double reciprocal 1/S Allosteric enzymes • Why the sigmoid shape? • Allosteric enzymes are multi-subunit enzymes, each with an active site. • They show a cooperative response to substrates hyperbolic curve michaelis-menten kinetics Sigmoidal curve Irreversible Inhibition = Enzyme stops working permanently 1. 2. Destruction of enzyme Irreversible Inhibitor = forms covalent bonds to E (inactive E) Examples: – Diisopropylfluorophosphate • • – Cyanide and sulfide • • – Inhibit cytochrome oxidase bind to the iron atom Fluorouracil • – inhibits acetylcholine esterase binds irreversibly to –OH of serine residue inhibits thymidine synthase (suicide inhibition - metabolic product is toxic ) Aspirin • • Inhibits prostaglandin synthase acylates an amino group of the cyclooxygenase 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. The Effects of Enzyme Inhibitors Example • When a slice of apple is exposed to air, it quickly turns brown. This is because the enzyme odiphenyl 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. 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 20.8 31.7 50.0 Effect of para-hydroxybenzoic acid (PHBA) Tube Tube Tube Tube A B C D Tube A Tube B Tube C Tube D [S] 1/[S] 4.8 mM 1.2 mM 0.6 mM 0.3 mM 0.21 0.83 1.67 3.33 ( 0.040 0.024 0.016 0.010 Vi) 1/Vi 4.8 mM 1.2 mM 0.6 mM 0.3 mM 1/[S] 0.21 0.83 1.67 3.33 0.06 0 0.03 2 0.01 9 0.01 1 16.7 31.3 52.6 90.9 ΔOD ΔOD54 0 [S] 25 41 62 Effect of phenylthiourea 102 540 (Vi) 1/Vi I- Competitive Inhibition EI S Competitive V [S] v max Km [S] CI V [S] v max K m [S] Kic S+E + I E 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, 0.5Vmax +CI 0.5V max 3 2 Km Kmapp 1 -1/Km Km +CI 2 1/v, /µmol/min /µmol/min 1/v, [I] 1 Kic 1.5 K /Vmax Kmm /Vmax 1 1/Vmax 1/V app -1/Km -1/K m Kmapp/Vmax 0.5 No I max 0 0 0 10 20 30 [S], mM 40 50 -0.6 -0.4 -0.2 0 0.2 0.4 0.6 0.8 1/[S], /mM 1 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 E+P 1 K m 1 ' v Vmax [S] Vmax 2.5 55 44 0.5Vmax 33 + NCImax 0.5V 0.5Vmax 22 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/Km -1/K m Km 1.5 1/Vmaxapp Km/Vmaxapp + NC I 1 0.5 K K/V /V m mmax max 1/V max 1/Vmax 0 00 00 10 10 20 20 30 30 [S], [S],mM mM 40 40 50 50 -0.6 -0.4 -0.2 0 0.2 0.4 0.6 0.8 1/[S], /mM 1 No I 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 v, µmol/min 0.5Vmax + UC I 0.5V max 3 Km 2 0.5Vmax [I] ' 1 Kiu app Km 1 -1/Km Km -1/Km -1/K app m /µmol/min 1/v, /µmol/min 2 1.5 1/Vmaxapp Kmapp/Vmaxapp 1 0.5 1/Vmax Km/Vmax + UC I Km/Vmax No I 1/Vmax 0 0 0 10 20 30 [S], mM [S]. 40 50 -0.6 -0.4 -0.2 0 0.2 0.4 0.6 0.8 1/[S], 1/[S]. /mM 1 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. 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 Useful enzymes for early diagnosis of dental caries and periodontal disease 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. Isoenzymes of Creatine kinase • CK has 3 forms dimer B and M chains: • CK1= BB • CK2= MB • CK3=MM • Heart only tissue rich in CK2, increases 4-8 hr after chest painspeaks at 24 hr. • LDH peaks 2-3 days after MI. • New markers: Troponin T, Troponin I 5- Regulation (Effectors) Effectors can be classified: 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 • Irreversible • Reversible – Competitive – Non-competitive – Uncompetitive Increase or decrease in enzyme reaction rate is reflected in the graph of V versus S 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. Regulation of “Enzyme activity” A. Regulation at trascription level (slowest) B. Isozymes: Regulation specific to 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 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 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 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 but the release of these suicide enzymes during apoptosis is an on/off switch than a true regulation. 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 (Proinsulin Insulin) an on/off switch more than regulation. 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) 1- Control of [S] • Concentration of substrate and product also control the rate of reaction, providing a biofeedback mechanism. • Usually, 0.1Km<[SPhysiology]<10km Mild changes in [S] Change in enzyme activity Homotropic effectors – substrate itself (binding at different site than the active site) affects enzyme activity on other substrate molecules. Most often this is a positive effector. 2- Product inhibition • Enzyme is reversibly inhibited by the product. Example: hexokinase - first reaction in glycolysis, hexokinase 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. 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. 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. 4. Inhibition at a ramification of a biosynthesis pathway (sequential inhibition) 4- Positive Feedforward control • Earlier reactants in a metabolic sequence feedforward 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. 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 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. AP Uni Uni AP+Q Uni Bi A+BP+Q Bi Bi A+B+CP+Q+R+S Ter Quad 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). Random sequential (example) Ordered sequential (example) Ping pong or double displacement mechanism Double displacement (example) Other enzymes • Some ribonucleoprotein enzymes have been discovered. – The catalytic activity is in the RNA part. – They are called Ribozymes • Catalytic antibodies are called Abzymes. • In competitive inhibition the inhibitor is similar in structure to the substrate and binds to the enzyme at the active site, preventing the substrate from binding. In feedback inhibition, the inhibitor binds to the enzyme at a site away from the active site and acts by altering the shape of the enzyme in such a way that it is incapable of catalyzing the reaction. Feedback inhibition is a natural part of the process by which an organism regulates the chemical reactions that take place in its cells. In that sense it is done on purpose. Competitive inhibition usually involves inhibitors, commonly called poisons, that do not belong in the cell. • Enzymes may be regulated by 1.. 2.Competitive product inhibition and allosteric regulation (fastest). • Many enzymes are inhibited by either their products, or by other chemicals, often those from further down a metabolic pathway. • Such enzymes may be 'gatekeepers' to a specific branch of metabolism, Biochemical reaction When the chemical reaction occurs in a biological system it is called a biochemical reaction. Biological system: Mild conditions Simultaneous presence of different substances Specific needs in specific times Catalyst A Reactant B Product What are the biocatalysts ( Enzymes)? Proteins & RNA ??? Basic enzyme reaction Catalyst A Product Reactant Enzyme S Substrate S+E ES P Product ES (Enzyme-substrate complex) P+E k1 S B E k-1 Binding step S E k2 P Catalytic step