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Catalysis Lecture 12 Biocatalysis •Introduction to biocatalysis: basic consepts, terms •Biotransformations in industry Enzyme structure • Enzymes are proteins • They have a globular shape • A complex 3-D structure Human pancreatic amylase © Dr. Anjuman Begum The active site © H.PELLETIER, M.R.SAWAYA ProNuC Database • One part of an enzyme, the active site (active center), is particularly important • The shape and the chemical environment inside the active site permits a chemical reaction to proceed more easily Protein chain Substrate molecule Essential groups outside the active center + - Catalytic group Active center Binding group The active site Cofactors • An additional nonprotein molecule that is needed by some enzymes to help the reaction • Tightly bound cofactors are called prosthetic groups • Cofactors that are bound and released easily are called coenzymes • Many vitamins are coenzymes Nitrogenase enzyme with Fe, Mo and ADP cofactors Jmol from a RCSB PDB file © 2007 Steve Cook H.SCHINDELIN, C.KISKER, J.L.SCHLESSMAN, J.B.HOWARD, D.C.REES STRUCTURE OF ADP X ALF4(-)-STABILIZED NITROGENASE COMPLEX AND ITS IMPLICATIONS FOR SIGNAL TRANSDUCTION; NATURE 387:370 (1997) The substrate • The substrate of an enzyme are the reactants that are activated by the enzyme • Enzymes are specific to their substrates • The specificity is determined by the active site The Lock and Key Model • Fit between the substrate and the active site of the enzyme is exact • Like a key fits into a lock very precisely • The key is analogous to the enzyme and the substrate analogous to the lock. • Temporary structure called the enzyme-substrate complex formed • Products have a different shape from the substrate • Once formed, they are released from the active site • Leaving it free to become attached to another substrate The Lock and Key Model S E E E Enzymesubstrate complex Enzyme may be used again P P Reaction coordinate The Lock and Key Model • This explains enzyme specificity • This explains the loss of activity when enzymes denature The Induced Fit Model • Some proteins can change their shape (conformation) • When a substrate combines with an enzyme, it induces a change in the enzyme’s conformation • Making the chemical environment suitable for the reaction • The bonds of the substrate are stretched to make the reaction easier (lowers activation energy) • This explains the enzymes that can react with a range of substrates of similar types Induced-fit model The binding induces conformational changes of both E and S, forcing them to get a perfect match. Factors affecting Enzymes • • • • substrate concentration pH temperature inhibitors Substrate concentration: Non-enzymic reactions Reaction velocity Substrate concentration • The increase in velocity is proportional to the substrate concentration Substrate concentration: Enzymic reactions Vmax Reaction velocity Substrate concentration • Faster reaction but it reaches a saturation point when all the enzyme molecules are occupied. • If you alter the concentration of the enzyme then Vmax will change too. The effect of pH Optimum pH values Enzyme activity Trypsin Pepsin 1 3 5 7 9 11 pH Pepsin, Trypsin, - protease found in the digestive system , where they hydrolyses proteins The effect of pH • Extreme pH levels will produce denaturation • The structure of the enzyme is changed • The active site is distorted and the substrate molecules will no longer fit in it • At pH values slightly different from the enzyme’s optimum value, small changes in the charges of the enzyme and it’s substrate molecules will occur • This change in ionisation will affect the binding of the substrate with the active site. The effect of temperature • Q10 (the temperature coefficient) = the increase in reaction rate with a 10°C rise in temperature. • For chemical reactions the Q10 = 2 to 3 (the rate of the reaction doubles or triples with every 10°C rise in temperature) • Enzyme-controlled reactions follow this rule as they are chemical reactions • BUT at high temperatures proteins denature • The optimum temperature for an enzyme controlled reaction will be a balance between the Q10 and denaturation. The effect of temperature Q10 Enzyme activity 0 10 20 30 40 Temperature / °C Denaturation 50 The effect of temperature • For most enzymes the optimum temperature is about 30°C • Many are a lot lower, cold water fish will die at 30°C, because their enzymes denature • A few bacteria have enzymes that can withstand very high temperatures up to 100°C • Most enzymes however are fully denatured at 70°C Inhibitors • Inhibitors are chemicals that reduce the rate of enzymic reactions. • The are usually specific and they work at low concentrations. • They block the enzyme but they do not usually destroy it. • Many drugs and poisons are inhibitors of enzymes in the nervous system. • They have nothing to do with negative catalysis The effect of enzyme inhibition • Irreversible inhibitors: Combine with the functional groups of the amino acids in the active site, irreversibly. Examples: nerve gases and pesticides, containing organophosphorus, combine with serine residues in the enzyme acetylcholine esterase. Pesticide poisoning choline esterase acetylcholine choline + acetic acid Acetylcholine accumulation will cause excitement of the parasympathetic system: vomitting, sweating, muscle trembling, pupil contraction + E OH P R'O RO X RO + P O organophosphate O E R'O AChE O acid inhibited AChE + N HX CHNOH CH3 E OH PAM O OR' P + N CH3 CHNO OR Heavy metal poisoning • Heavy metal containing chemicals bind to the –SH groups to inactivate the enzymes. S SH + E Hg2+ SH 2H+ S Cl + E + S SH SH Hg E Cl As C H CHCl E S As C H CHCl + 2HCl The effect of enzyme inhibition • Reversible inhibitors: These can be washed out of the solution of enzyme by dialysis. There are two categories. The effect of enzyme inhibition 1. Competitive: These compete with the substrate molecules for the active site. The inhibitor’s action is proportional to its concentration. Resembles the substrate’s structure closely. E+I Reversible reaction EI Enzyme inhibitor complex The effect of enzyme inhibition Fumarate + 2H++ 2e- Succinate Succinate dehydrogenase CH2COOH COOH CHCOOH CH2 CH2COOH COOH Malonate CHCOOH The effect of enzyme inhibition 2. Non-competitive: These are not influenced by the concentration of the substrate. It inhibits by binding irreversibly to the enzyme but not at the active site. Examples • Cyanide combines with the Iron in the enzymes cytochrome oxidase. • Heavy metals, Ag or Hg, combine with –SH groups. These can be removed by using a chelating agent such as EDTA. Applications of inhibitors • Negative feedback: end point or end product inhibition • Poisons snake bite, plant alkaloids and nerve gases. • Medicine antibiotics, sulphonamides, sedatives and stimulants Enzymes – key elements for biocatalysis Enzyme class Catalyzed reaction Oxidoreductases Oxidation-reduction reaction Transferases Transfer of functional group Hydrolases Hydrolytic reactions Lyases Group elimination (forming double bonds) Isomerizaion reaction Isomerases Ligases Bond formation coupled with a triphosphate cleavage Accelerated reaction rates Non-enzymatic rate constant (kn in s-1) Enzymatic rate constant (kn in s-1) 10-1 106 Chymotrypsin 4 x 10-9 4 x 10-2 Lysozyme 3 x 10-9 5 x 10-1 Triose phosphate isomerase 4 x 10-6 4 x 103 Urease 3 x 10-10 3 x 104 Mandelate racemase 3 x 10-10 5 x 102 Alkaline phosphatase 10-15 102 Enzyme Carbonic anhydrase Kinetics Reaction rate [P] [P] Initial slope = vo = t 0 Time (t) [P] t Initial velocity • The reaction rate is defined as the product formation per unit time. • The slope of product concentration ([P]) against the time in a graphic representation is called initial velocity. • It is of rectangular hyperbolic shape. Reaction velocity curve V0 Vmax Vmax/2 [S] 0 Km Intermediate state Forming an enzyme-substrate complex, a transition state, is a key step in the catalytic reaction. k1 E + S ES k3 E + P k2 initial intermediate final Free energy transition state, S G+ (uncatalyzed) G+ (catalyzed) reactants G for the reaction products Reaction progress Rate constants k1 E + S ES k3 E + P k2 • k1 = rate constant for ES formation • k2 = rate constant for ES dissociation • k3 = rate constant for the product released from the active site Michaelis-Menten Equation • The mathematical expression of the product formation with respect to the experimental parameters • Michaelis-Menten equation describes the relationship between the reaction rate and substrate concentration [S]. Assumptions • [S] >> [E], changes of [S] is negligible. • k2 is negligible compared with k1. • Steady-state: the rate of E-S complex formation is equal to the rate of its disassociation (backward E + S and forward to E + P) [S] V = K V max m + [S] Describing a hyperbolic curve. Km is a characteristic constant of E [S] << Km ,v ∝ [S] [S] >> Km ,v ≈ Vmax V0 Vmax Zero order with respect to [S] First order with respect to [S] 0 [S] Significance of Km • the substrate concentration at which enzyme-catalyzed reaction proceeds at onehalf of its maximum velocity • Km is independent of [E]. It is determined by the structure of E, the substrate and environmental conditions (pH, T, ionic strength, …) V0 Vmax Vmax/2 [S] 0 Km • Km is a characteristic constant of E. • The value of Km quantifies the affinity of the enzyme and the substrate under the condition of k3 << k2. The larger the Km, the smaller the affinity. k2 + k3 Km = k1 Km for selected enzymes Enzyme Substrate km Catalase H2O2 25 Hexokinase ATP 0.4 D-Glucose 0.05 D-Fructose 1.5 Carbonic anhydrase HCO3- Chemotrypsin Glycyltyrosinylglycine 108 N-Benzoyltyrosinamide 2.5 9 Galactosidase D-Lactose 4 Threonine dehydratase L-Threonine 5 Significance of Vmax • The reaction velocity of an enzymatic reaction when the binding sites of E are saturated with substrates. • It is proportional to [E]. Turnover number k3 = Vmax / [E] • Vmax is the reaction rate when the enzymes are saturated, and is independent of the enzyme concentration. • The number of the products converted in a unit time by one enzyme molecule which is saturated. Lineweaver-Burk plot Km 1 1 1 + = Vmax [S] Vmax V • To determine Km and Vmax • To identify the reversible repression Double-reciprocal plot 1/V Slope = Km/Vmax Intercept = -1/ Km Intercept = 1/Vmax 1/[S] Enzyme activity • Determination of the enzymatic activity requires proper treatment of enzymes, excess amount of substrate, optimal T and pH, … • One katal is the amount of enzyme that converts 1 mol of substrate per second. • IU = 16.67×10-9 kat Fermentation and Biotransformation Enzymatic processes Examples Fermentation of glucose to alcohol Yeast metabolize glucose for their metabolic processes Anaerobic glycolysis – Production of 2 equivalents of ATP for cells energy per equivalent of glucose metabolized While this process is underway, two enzymatic pathways are coupled that use/regenerate NAD – the cofactor in the enzyme – alcohol dehydrogenase O O O O NADH + H+ NAD+ + O CH3 pyruvate HC H2C O CH3 acetaldehyde alcohol dehydrogenase CH3 ethanol OH Chemistry of Alcohol Dehydrogenase A. Reduction of acetaldehyde • Normally in the metabolic process of yeast, acetaldehyde is reduced to ethanol by the addition of hydrogen across the carbon-oxygen double bond by the enzyme NADH + H+ NAD+ H H H3C H3C C O acetaldehyde • alchohol dehydrogenase C H O H ethanol This reaction is very similar to the sodium borohydride reduction of aldehydes and ketones H H H3C O NaBH4 R-OH H3C H O H acetaldehyde ethanol B. Enzymatic Reduction 1. Hydride source Within the enzyme, the hydride source is nicotinamide dinucleotide (NAD/H): O H O H H C C NH2 NH2 O - O P O N O - O H H OH H H O H P O N O H H OH H H + H O H H OH H H reduced nicotinamide dinucleotide, NADH H P N ON O hydride NH2 N O N N O O N - O P H NH2 N O O N O H H OH H H H nicotinamide dinucleotide, NAD+ In biological systems, a reagent such as NaBH4 (chemical reduction) is too strong, and the donation of hydride is NOT reversible Once more NaBH4 reacts with water to produce hydrogen gas so it cannot exist long or be regenerated in a living system From NADH to NAD+, the donation of hydride is an equilibrium process (controlled by Le Chatlier’s principle), as both the oxidized (NAD+) and reduced (NADH) forms are roughly equal in energy The equilibrium constant for this process under biological conditions is close to one. The reasons for come from the stability of both forms of NAD: H O H O H C C NH2 - H NH2 N N R R + H hydride NAD+ NADH + H H Large concentration of positive charge density in the contributing resonance structure favors reverse reaction. O C NH2 N R Contributing resonance structure Hydride donation leads to formation of an aromatic ring, Favoring the forward reaction Mechanism of reduction (acetaldehyde) H O H3C Base O Base H H3C H H H O H O H H C C NH2 NH2 N N R R NADH NAD+ Of course in biological systems, the reverse reaction also occurs: O Base H H3C H O H3C H H H O O H H H C C NH2 NH2 N N R R NAD+ NADH In fact, the enzyme itself is named for this reverse reaction: alcohol dehydrogenase Base 2. Active site of the Enzyme: Unlike the NaBH4 reduction, this reaction is stereospecific Active site of enzyme must provide three point recognition of the substrate Active site must also provide the the second hydrogen, as H+ to complete the reduction of the C=O in the forward reaction and accept H+ in the reverse reaction The active site of alcohol dehydrogenase Histidine 67 H N Acetaldehyde Enters from the bottom N Cysteine 48 H2 C Zn+2 S S H2 C Cysteine 174 O H O H2 C Serine 48 O H3C H3C C H C Recognition occurs through the Zn+ atom, the protonated base, and NADH H O H H C NH2 N O R H3C C NADH H H H Hydride is added from Upper face of acetaldehyde Antibiotics Penicillin and 6-Aminopenicillanic Acid (6-APA) Penicillin: First discovered by Fleming in 1932 19% of worldwide antibiotic market. Superior inhibitory action on bacterial cell wall synthesis Broad spectrum of antibacterial activity Low toxicity Outstanding efficacy against various bacterial strains Excessive use has led to development of resistant pathogens 6-APA: Raw material for production of new semisynthetic penicillins (amoxycillin and ampicillin) Fewer side effects Diminished toxicity Greater selectivity against pathogens Broader antimicrobial range Improved pharmacological properties Chemical and Enzymatic Deacylation of Penicillins to 6-APA R C H N S O N CH3 CH3 Penicillin acylase COOH O Penicillin V or G [R=Ph or PhO] S NH2 Alkaline [Enzymatic] N O (6-APA) R PCl5 ROH H2O [Chemical] Pyridine Me3SiCl C H N S O N O CH3 CH3 COOSiMe3 CH3 CH3 COOH Penicillin and 6-Aminopenicillanic Acid (6-APA) Chemical method: Use of hazardous chemicals - pyridine, phosphorous pentachloride, nitrosyl chloride Enzymatic method: Regio- and stereo-specific Mild reaction conditions (pH 7.5, 37 oC) Enzymatatic process is cheaper by 10% Enzymes: Penicillin G acylase (PGA)- Escherichia coli, Bacillus megaterium, Streptomyces lavendulae Penicillin V acylases (PVA)- Beijerinckia indica var. Penicillium, Fusarium sp., Pseudomonas cidovorans Immobilized Enzyme: Life, 500-2880 hours Aspartame (L-Asp-L-Phe-Methyl Ester) Aspartame is dipeptide sweetener formed by linking the methyl ester of phenylalanine with aspartic acid Extensively used in food and beverages 200 times as sweet as sucrose Annual sale: 200 million kg, $ 850 million Nutrasweet Corp. retains 75% of the US market Chemical method: The amino group of aspartic acid needs to be protected to prevent its reacting with another molecule of aspartic acid to give unwanted by-products The correct single enantiomer of each of the reactants must be used to give the required stereochemistry of aspartame (beta-aspartame is bitter tasting) Enzymatic method: Thermolysin promotes reaction only at the alpha-functionality Mild condition, pH 6-8, 40 oC Biocatalytic Production of Aspartame HO2C Ph + PhCH2OCNH CO2H O N-Cbz-aspartic acid H2N CO2Me D,L-phenylalanine Methyl ester thermolysin H2O HO2C PhCH2OCNH Cbz, benzyloxycarbonyl O Ph CNH CO2Me O Cbz-aspartame Important Factors in Using Enzymes • Reactions possible that are not possible using chemistry • Specificity of reaction including substrate specificity, positional specificity, stereospecificity • Allows milder process conditions e.g. temperature, pH, sterility etc. • Reduces number of process steps required • Eliminates the need to use organic solvents in processing • Immobilization of enzyme to allow its reuse or continuous use • Use of enzymes in combination with other separate chemical steps • Genetic engineering to improve enzymes Environmentally Compatible Synthesis of Catechol from Glucose acetone a hydroquinone b HO benzene cumene CO2H OH O HO CO2H OH d OH OH D-glucose (a) (b) (c) (d) phenol OH HO d d O c OH catechol HO OH OH 3-dehydroshikimic protocatechuic acid acid propylene, solid H3PO4 catalyst, 200-260°C, 400-600 psi. O2, 80-130°C then SO2, 60-100°C. 70% H2O2, EDTA, Fe2+ or Co2, 70-80°C. E. coli AB2834/pKD136/pKD9.069A, 37°C. Draths and Frost, 1995 Biological function of some metals Na, K Charge transfer, ostmotic equilibrium Mg Structure, Hydrolase, Isomerase Ca Structure, charge transfer, releaser V Oxidase, N2 binding Cr Unknown – glucose tolerance Mo Oxidase, nitrogen binding, oxo gruop transfer W Dehydrogenase Mn Photosynthesis, oxidase, structure Fe Oxidase, O2 transport and storing, electron transfer, N2 binding Co Oxidase, alkyl group transfer Ni Hydrogenase, hydrolase Cu Oxidase, O2 transport, electron transfer Zn Structure, hydrolase Catalysis by Carbonic anhydrase (carbonate dehydratase) H O_ H+ H Zn His94 His119 His96 H CO2 O His94 His96 O Zn His119 C H O_ HCO3- O Zn His94 His119 His96 H2O H O O O _ O Zn His94 His119 His96 H _ O O Zn His94 His119 His96 Industrial Enzyme Market Annual Sales: $ 1.6 billion Food and starch processing: Detergents: Textiles: Leather: Pulp and paper: 45% 34% 11% 3% 1.2% Biotransformations in industy References • P. Billiet „Enzymes” • http://202.118.40.5/biochemistry/ewebeditor/uploadfile/20091117093812 957.ppt