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The Chemical Reactions of Life

The Chemical Reactions of Life
UNIVERSITY OF OXFORD DEPARTMENT OF CHEMISTRY Basic Enzymology Dr Emily Flashman http://flashman.chem.ox.ac.uk 2 Lectures – Trinity Term 2015 Enzymology: Lecture 1 Topics to be Covered: Lecture 1: Introduction to enzymes The importance of enzymes in catalysing the chemical reactions of life. How enzymes promote catalysis Typical enzymecatalysed reactions Enzyme efficiency: selectivity, cofactors and control. Lecture 2: Principles of enzyme catalysis Thermodynamics of enzymecatalysed reactions Transition state stabilisation Case Study Triose phosphate isomerase Enzymology: Lecture 1 Topics to be Covered: No prior knowledge other than content of Oxford Chemistry course. No text books are essential but the following are useful: Foundations of Chemical Biology, Oxford, Chemistry Primer (Dobson, Gerrard, Pratt, OUP) Access to a modern Biochemistry textbook: Voet and Voet, Biochemistry (4th Edition, Wiley) Enzymology: Lecture 1 Objectives of the Course: • To provide an introduction to enzyme catalysis and related protein functions, focusing on chemical principles • To connect mechanistic synthetic chemistry with biology • To demonstrate that chemical principles underlie biology and that understanding and manipulating this chemistry is fascinating and the basis of multiple applications. Enzymology: Lecture 1 This course is NOT about remembering complex structures But About understanding the chemical principles that control biology "Enzyme catalysis: not different just better"(Jeremy Knowles) Enzymology: Lecture 1 • One of the most important targets for pharmaceuticals • Intrinsic in human biology manipulation • Fermentation of pharmaceuticals and fine chemical • Useful in synthesis, especially for resolutions / asymmetric reactions, e.g. resolutions by enantiospecific amino acid acylase • Wide ranging applications in society, e.g. food industry, washing powders • Bioremediation • An understanding of enzyme catalysed reactions may help us to design useful unnatural ‘biomimetic’ catalysts, based on knowledge of enzyme structures / mechanisms. The Chemical Reactions of Life Enzymology: Lecture 1 The Chemical Reactions of Life Enzymology: Lecture 1 The Chemical Reactions of Life Enzymology: Lecture 1 Hexokinase creates right conditions for nucleophilic attack of C6 OH on ATP The enzyme promotes an otherwise unfavourable reaction that is vital in energygenerating glycolysis process. The Chemical Reactions of Life Enzymology: Lecture 1 Enzymes are important How Enzymes Promote Catalysis Enzymology Lecture 1 Enzymes are proteinbased catalysts – they facilitate chemistry • ‘Free enzymes’ are often globular proteins, but enzymes can be part of large complexes or embedded in membranes. • We will focus on ‘simple’ enzymes that catalyse ‘simple’ reactions, but the same principles of catalysis apply in all cases. How Enzymes Promote Catalysis Enzymology Lecture 1 Bringing enzyme and substrate(s) together in a favourable conformation to promote the reaction. Enzymes have ACTIVE SITES • a 3D cleft or crevice with precisely defined arrangement of atoms • relatively small area of enzyme • substrates bind via multiple weak interactions How Enzymes Promote Catalysis Enzymology Lecture 1 Enzymes are biological catalysts: Increase the rate at which a reaction reaches equilibrium Stabilise the transition state of a reaction relative to the uncatalysed reaction Enzymes are finely tuned for specificity in substrate binding and optimal arrangement of catalytic groups How Enzymes Promote Catalysis Enzymology Lecture 1 Amino acid side chains (and the peptide backbone) provide a repertoire of functional groups for catalysis and binding How Enzymes Promote Catalysis Enzymology Lecture 1 Enzymes are more efficient than chemical catalysts: 1. Higher reaction rates – by several orders of magnitude 2. Milder reaction conditions – low temps, atm pressure, neutral pH 3. Greater reaction specificity – no side products 4. Capacity for control – catalytic activity can vary in response to local conditions Typical EnzymeCatalysed Reactions Enzymology Lecture 1 Enzymes catalyse both simple reactions and reactions that are ‘impossible’ for synthetic chemistry. Triose phosphate isomerase (‘easy’ reaction) Proline hydroxylase (‘difficult’ reaction) Typical EnzymeCatalysed Reactions Enzymology Lecture 1 Penicillin biosynthesis catalysed by isopenicillin N synthase • Fermented on a tonscale by fermentation • Fermented pencillins are used directly and others are produced by modification of fermented penicillins • Single step reaction into highly functionalised penicillin • Organic synthesis is not competitive (1 multistep route) Typical EnzymeCatalysed Reactions Enzymology Lecture 1 Enzymes catalyse (most) of the ‘fundamental’ reactions of organic synthesis Example 1 – the S 2 reaction by a methyltransferase enzyme NTypical EnzymeCatalysed Reactions Enzymology Lecture 1 Example 2 – the Michael reaction (conjugate addition) by enoyl CoA hydratase Enzyme Efficiency Enzym Enzym olo olog gy y Lect Lect uru e re 1 1 How do enzymes manage to be such efficient catalysts 1. Substrate specificity • Stereospecificity • Geometric specificity 2. Coenzymes 3. Control of activity: regulating activity in time and space Enzyme Efficiency: Substrate Specificity Enzymology Lecture 1 Substrates interact with enzymes via van der Waals, electrostatic, hydrogen bonding and hydrophobic interactions Induced fit model Lock and key model Substrates have geometric and electronic complementarity with their binding site on the enzyme Enzyme Efficiency: Substrate Specificity Enzymology Lecture 1 Enzymes consist of naturallyoccurring Lamino acids = they form assymetric active sites and only catalyse reactions with substrates with complementary chirality e.g. hexokinase only catalyses phosphorylation of Dglucose, chymotrypsin only catalyses hydrolysis of Lamino acids. Enzymes can be used to resolve racemic mixtures of compounds, e.g. Nacyl amino acids Enzyme Efficiency: Substrate Specificity Enzymology Lecture 1 Most enzymes are selective about the chemical groups that will fit into their active sites Enzymes vary in their degree of specificity Most enzymes catalyse the reaction of a small range of related reactions e.g. yeast alcohol dehydrogenase (YADH) catalyses oxidation of small primary and secondary alcohols, but ethanol is most efficient Enzyme Efficiency: Cofactors Enzymology Lecture 1 Enzymes are good at acid/base reactions, transient covalent bonds and charge charge interactions Not so good at redox reactions and group transfer processes Need Cofactors Apoenzyme (inactive) + cofactor Metal ions Organic molecules 2+ e.g. Fe Holoenzyme (active) Transient Prosthetic group + e.g. NAD e.g. FAD, haem; (usually); redox redox Enzyme Efficiency: Cofactors Enzymology Lecture 1 Metal cofactors are often transition metals Cofactor Example I – Complexation with Zn(II) reduces the pKa of alcohols/ water in amide hydrolysis reactions (metalloproteases are common) Cofactor Example II Transition metals are used to activate triplet state dioxygen, e.g. superoxide dismutase Enzyme Efficiency: Substrate Specificity Enzymology Lecture 1 Cofactor Example III The cofactor NADH is a biological equivalent of NaBH 4 Note the Stereoselective Htransfer which H is lost depends on the enzyme. This chemistry is used in ethanol metabolism. Enzymes: Control of Enzyme Activity Enzymology Lecture 1 Necessary to coordinate metabolic processes, respond to changes in environment, grow and differentiate And to prevent inappropriate catalysis – enzymes are powerful catalysts and need to be regulated Enzymes need to be in the right place at the right time Control of Enzyme Availability • Rate of enzyme synthesis (genetic control of expression) • Rate of enzyme degradation • Compartmentalisation Enzymes: Control of Enzyme Activity Enzymology Lecture 1 Control of Enzyme Activity – structural or conformational alterations • Activation by cleavage of inactive proenzymes (e.g. in serine proteases) • Requirement for cofactors/cosubstrates • Activation by post translational modifications (of enzyme or of protein substrates) • Inhibition by small molecules (feedback inhibition is important in metabolism) an enzyme is only as active as the amount of enzyme:substrate complex inhibition direct allosteric e.g. product inhibition Enzymes: Classifications Enzymology Lecture 1 1.Oxidoreductases Oxidation and reduction Dehydrogenase,reductase,o transfer of electrons xidase and oxygenases 2. Transferases Transfer of function groups Acetyltransferase, methyl eg. Acetyl, methyl and transferase, protein kinase phosphate and polymerase 3. Hydrolases Hydrolysis reactions where a Protease, nuclease and molecule is split by the phosphatase addition of water 4. Lyases Catalyze the cleavage of CC, Decarboxylase and aldolase CO and CN bonds ( not hydrolysis or oxidation) 5. Isomerases Atomic rearrangement within Racemase and mutase molecules 6. Ligases Join the two molecules DNA ligase, peptide together (using ATP) synthase, fatty acid synthase http://www.chem.qmul.ac.uk/iubmb/enzyme Enzymes: A couple of other points… Enzymology Lecture 1 • Enzymes are not the only biological catalysts – Ribosomes are made of rRNA and catalyse protein synthesis + Many reactions in cells are probably catalysed by ions (H ) • Enzymes do not only catalyse covalent reactions but also noncovalent 'processes' / conformational changes’ e.g. chaperones catalyse protein folding and cistrans prolylamide bond isomerases. O O O R R N H R N R N O H R cis trans prolineEnzymes: Lecture 1 Summary Enzymology Lecture 1 Enzymes are amazing • Superefficient catalysts • Catalyse reactions not possible synthetically • Precise 3D arrangement of amino acids at active site and beyond • Highly specific • Often require cofactors • Activity must be controlled and regulated How do they do it Enzym Enzym ology Lect ology ur : e Lect 2 ure 1 Topics to be Covered: Lecture 1: Introduction to enzymes The importance of enzymes in catalysing the chemical reactions of life. How enzymes promote catalysis Typical enzymecatalysed reactions Enzyme efficiency: selectivity, cofactors and control. Lecture 2: Principles of enzyme catalysis Thermodynamics of enzymecatalysed reactions Transition state stabilisation Case Study Triose phosphate isomerase Enzymes and Thermodynamics Enzym Enzym ology Lect ology ur : e Lect 2 ure 1 Topics to be Covered: Enzymes and Thermodynamics Enzymology Lecture 2 ΔG = free energy change of a reaction Negative ΔG  spontaneous reaction ΔG is energy difference between reactants and products ΔG reveals nothing about rate of reaction ΔG reveals nothing about mechanism of reaction Equilibrium constant CD  GRT lnK eq GGRT ln AB At equilibrium, ΔG = 0 K' depends on the concentration of A eq B C D ΔG therefore dependent on substrate concentration Enzymes and Thermodynamics Enzymology Lecture 2 An enzyme cannot alter the equilibrium of a chemical reaction The amount of product formed will be the same whether or not the enzyme is present The difference is the rate at which equilibrium is reached and the rate of product formation (seconds compared to hours) Pathway from substrate to product So how do enzymes increase the RATE of a reaction Enzymes and Thermodynamics Enzymology Lecture 2 ‡ A reaction must go through a ΔG transition state (high energy) S before product formation P ‡ Activation energy (ΔG ) is that required to reach the transition state Enzymes and Thermodynamics Enzymology Lecture 2 Linus Pauling "An enzyme stabilises the transition state of the catalyzed reaction more than it stabilises the substrate and the product" Enzymes and Thermodynamics Enzymology Lecture 2 If the ES complex, transition state and EP complex are all stabilised by the same amount then: ‡ Equal stabilisation results in no change in ΔG therefore no catalysis.Enzymes and Thermodynamics Enzymology Lecture 2 However, if the ES and EP complexes are destabilised relative to the transition state and/or transition state sufficiently stabilised: ‡ Binding energy is ‘used’ to lower ΔG . ES and EP are not significantly stabilised relative to E+S and E+P. Enzymes and Thermodynamics Enzymology Lecture 2 Enzymology: Lecture 2 Transition state stabilisation How do enzymes achieve transition state stabilisation Bringing enzymes and substrates together in a favourable conformation to promote formation of the transition state. Substrate binds to enzyme active site • a 3D cleft or crevice with precisely defined arrangement of atoms • relatively small area of enzyme • substrates bind via multiple weak interactions Enzymology: Lecture 2 Transition state stabilisation How do enzymes achieve transition state stabilisation 1. Reduction in entropy – intramolecular reactions Intramolecular reactions are faster than equivalent intermolecular reactions Note stereoelectronic geometry requirements must always be met. Enzymology: Lecture 2 Transition state stabilisation How do enzymes achieve transition state stabilisation 1. Reduction in entropy – intramolecular reactions 2. Optimal orientation of substrates 3. Binding energy provided by interaction between enzyme and substrate 4. Bond strain can be tolerated Example – In some proteases the 'oxyanion hole' polarises the pibond of the amide carbonyl making it more susceptible to nucleophilic attack. • There is good geometric fit of charge in the transition state Enzymology: Lecture 2 Transition state stabilisation Transition state stabilisation enables catalysis by: • Hydrophobic interactions • Electrostatic interactions • Acid/base interactions • Metal ion interactions • Covalent reactions with substrate (covalent catalysis) • Cofactors Case Study: Triose Phosphate Isomerase (TPI) Enzymology Lecture 2 • Catalyses the interconversion of Dglyceraldehyde3phosphate (GAP) and dihydroxyacetonephosphate (DHAP) • Ketoenol tautomerisation • TPI (TIM) is amongst the best understood of all enzymes. Jeremy Knowles • Good illustration of transition state stabilisation DGAP DHAP Case Study: Triose Phosphate Isomerase (TPI) Enzymology Lecture 2 Uncatalysed reaction Enolate intermediate DHAP I S P DGAP Case Study: Triose Phosphate Isomerase (TPI) Enzymology Lecture 2 TPI Catalysed reaction TIM increases the rate of this 10 reaction by 10 DGAP DHAP E + S E.S I E.P E + P Stabilisation of intermediate = reduced ΔG to reach transition state Enolate intermediate tightly bound at TIM active site Case Study: Triose Phosphate Isomerase (TPI) Enzymology Lecture 2 • a/b barrel • Same motif found in other glycolytic enzymes, e.g. aldoloase, enolase • Glu 165 and His 95 essential for catalysis • Loop closes on substrate binding Case Study: Triose Phosphate Isomerase (TPI) Enzymology Lecture 2 • Important in GLYCOLYSIS • Glycolysis is the first (anaerobic) pathway in the conversion of glucose to ATP (energy) • At equilibrium, reaction favours DHAP, but GAP consumed so rapidly that reaction essentially flows in this direction Case Study: Triose Phosphate Isomerase (TPI) Enzymology Lecture 2 GAP GGRT ln  GRT lnK eq DHAP  G2.303RT log 10K eq At equilibrium, ratio of GAP:DHAP = 0.0475, i.e. Kˈ = 0.0475, eq under standard conditions.  G2.303RT log 10K eq  G7.53 kJ/mol endergonic 4 If DHAP is present in excess, e.g. 2 x 10 M compared to GAP at 3 x 6 10 M, then Kˈ changes and ΔG˚ˈ = 2.89 kJ/mol exergonic eq Reaction will only proceed when DHAP is in excess… Case Study: Triose Phosphate Isomerase (TPI) Enzymology Lecture 2 DGAP Enolate intermediate DHAP Transition states bind to enzymes more tightly than substrates Proof of involvement of enolate intermediate from the use of transition state analogues: Which bind more tightly than substrate, do not act as substrate, and inhibit TPI reaction Case Study: Triose Phosphate Isomerase (TPI) Enzymology Lecture 2 Transition states 3. Glu165 1. H abstraction stabilised by Hbonds protonates enediol by Glu165 GAP DHAP 2. Protonation 4. H abstraction of carbonyl O by His95 by His95 Case Study: Triose Phosphate Isomerase (TPI) Enzymology Lecture 2 Reaction likely occurs via concerted general acidbase catalysis • Due to lowbarrier hydrogen bonds formed when pK’s of Hbond acceptor and donor groups are near equal • Contribute to stabilisation of transition state GAP DHAP Case Study: Triose Phosphate Isomerase (TPI) Enzymology Lecture 2 Evidence for role of Glu165: 1. Affinity labelling reagents employed to identify base at active site of TPI: • Detect binding of inhibitor by (i) stoichiometry of radiolabelled compound, (ii) digestion of enzyme and identification of labelled fragment by mass spectrometry. Case Study: Triose Phosphate Isomerase (TPI) Enzymology Lecture 2 Evidence for Role of Glu165: 2. Mutagenesis, Xray crystallography and kinetics: Replace Glu165 with Asp Carboxylate group withdrawn by 1Å – catalytic efficiency reduced 1000 fold Case Study: Triose Phosphate Isomerase (TPI) Enzymology Lecture 2 Importance of the • Xray crystallography flexible loop: comparing E and E.S analogue • Loop acts like a hinged lid • Lysine residue on loop interacts with phosphate group on substrate • Loop helps stabilise transition state Case Study: Triose Phosphate Isomerase (TPI) Enzymology Lecture 2 In Solution: Conformation of phosphate group in plane of hydrogen being removed allows βelimination Methyl glyoxal toxic In Enzyme: Phosphate group held away from the plane, preventing βelimination and facilitating specific reaction Summary Enzymology Lecture 2 • Enzymes cannot promote reactions that are not otherwise thermodynamically favourable • Enzymes catalyse reactions by stabilising the transition state • Transition state is stabilised by very specific interactions at the enzyme active site • The wellcharacterised enzyme triose phosphate isomerase is a good example of transition state stabilisation by: o acidbase reactions o a stabilising loop to confer product selectivity Extra topic: Catalytic Antibodies Enzymology Lecture 2 Can we design a novel biological catalyst a complementary structure should catalyse the reaction Extra topic: Catalytic Antibodies Enzymology Lecture 2 Antibodies contain hypervariable regions that bind haptens Extra topic: Catalytic Antibodies Enzymology Lecture 2 If the Pauling theory of enzyme catalysis by stabilisation of transition states is correct, one way to design a de novo catalyst for a reaction would be to create a structure complementary to the transition state for the reaction. 1. Synthesis of transition state analogue = hapten 2. Link hapten to carrier protein to give haptenantigen 3. Immune system 4. Production of antibodies 5. Identification of catalytic antibodies Extra topic: Catalytic Antibodies Enzymology Lecture 2 3 5 But catalytic antibodies only enhance rates by 10 /10 (at 10 best) – enzymes 10 …. Why Enzymes are more than just rigid template for a single transition state • More than one transition state • Catalysis is not simple binding • Product/substrate inhibition need to be avoided • Orientation of substrate in important • Cofactors maybe are required • Antibody substructures are limited by their folds Catalytic antibodies do have therapeutic promise, e.g. Evac, an ‘abzyme’ against HIV (prevents invasion of host cells). www.abzymeresearchfoundation.org
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