2011.09.13.. TÁMOP –4.1.2-08/2/A/KMR-2009-0006 1 Development of Complex Curricula for Molecular Bionics and Infobionics Programs within a consortial* framework** Consortium leader PETER PAZMANY CATHOLIC UNIVERSITY Consortium members SEMMELWEIS UNIVERSITY, DIALOG CAMPUS PUBLISHER The Project has been realised with the support of the European Union and has been co-financed by the European Social Fund *** **Molekuláris bionika és Infobionika Szakok tananyagának komplex fejlesztése konzorciumi keretben ***A projekt az Európai Unió támogatásával, az Európai Szociális Alap társfinanszírozásával valósul meg. PETER PAZMANY CATHOLIC UNIVERSITY SEMMELWEIS UNIVERSITY dk_fejlec.gif INFOBLOKK ITK_logo_new_375_v1 2011.09.13.. TÁMOP –4.1.2-08/2/A/KMR-2009-0006 2 Semmelweis University ORGANIC AND BIOCHEMISTRY Thermodynamics and kinetics of metabolic pathways http://semmelweis-egyetem.hu/ (Szerves és biokémia) (Metabolikus utak termodinamikája és kinetikája) KrasimirKolev Biochemistry: Thermodynamics in metabolism 2011.09.13.. TÁMOP –4.1.2-08/2/A/KMR-2009-0006 3 Lecture objectives At the end of the presentation the participant will be able: 1) To define the subject of Biochemistry 2) To discuss the reductionist approach to understanding metabolism 3) To describe the connection between thermodynamics and directionality of metabolic pathways 4) To interpret the spontaneity of reactions in living organisms in terms of coupling-in-series and coupling-in-parallel of reactions 5) To define the terms near-equilibrium and non-equilibrium reactions 6) To understand the relation between thermodynamics and kinetics of metabolic pathways 7) To describe the structure of proteins 8) To list the basic steps in the catalytic mechanism of serine proteases 9) To discuss the structure/function relations in enzyme action 10) To interpret the action of protease inhibitors Biochemistry: Thermodynamics in metabolism 2011.09.13.. TÁMOP –4.1.2-08/2/A/KMR-2009-0006 4 Cell structure.jpg The subject of BiochemistryDEF: chemical transformations occurring in living organisms Specific features: • moderate temperature • standard pressure • compartments • open systems 2011.09.13.. TÁMOP –4.1.2-08/2/A/KMR-2009-0006 5 Biochemistry: Thermodynamicsin metabolism metabolicpathways_updated_02_07.jpg Biochemistry at a glance Metabolic charts http://www.iubmb-nicholson.org/ the road maps of chemical transformations in the cell Thermodynamics and kinetics the rules for substance fluxes (which one? when? where? how?) 2011.09.13.. TÁMOP –4.1.2-08/2/A/KMR-2009-0006 6 Biochemistry: Thermodynamics in metabolism potter.jpg The reductionist approachDEFto establish the rules 2011.09.13.. TÁMOP –4.1.2-08/2/A/KMR-2009-0006 7 Biochemistry: Thermodynamicsin metabolism Thermodynamic rules the energy transformations determine the direction and spontaneity of the processes Valid for: • a single reaction • a series of reactions (= metabolic pathway) • transport processes 2011.09.13.. TÁMOP –4.1.2-08/2/A/KMR-2009-0006 8 Biochemistry: Thermodynamics in metabolism The first law of thermodynamics (the law of energy conservation) enthalpyDEFchange heat work Reaction .H0(kJ/mol) palmiticacid+ 23O216CO2+16H2O -10024 glucose+ 6O26CO2+6H2O -2813 alanine+ 3O22.5CO2+2.5H2O + 0.5urea -1304 ATP+ H2OADP+Pi -21 2011.09.13.. TÁMOP –4.1.2-08/2/A/KMR-2009-0006 9 Biochemistry: Thermodynamics in metabolism Energy requirements of human subjects during different activities Intensity Activity Mean energy requirement(kJ/min) basal activity rest 4.5 low office work, driving 6 –10 moderate housekeeping, swimming 15 –25 high digging, marathon running 30 -85 2011.09.13.. TÁMOP –4.1.2-08/2/A/KMR-2009-0006 10 Biochemistry: Thermodynamics in metabolism Heat production in the living organisms glukóz + 6O2 6H2O + 6CO2 38ADP + 38Pi 38ATP + 38H2O .H=-2813 kJ .H=798 kJ the difference is released as heat 2011.09.13.. TÁMOP –4.1.2-08/2/A/KMR-2009-0006 11 Biochemistry: Thermodynamics in metabolism The second law of thermodynamics (for spontaneous processes .S>0) entropyDEF =heat absorbed in a reversible reaction/ temperature K heat temperature 2011.09.13.. TÁMOP –4.1.2-08/2/A/KMR-2009-0006 12 Biochemistry: Thermodynamics in metabolism Gibbs’ free energyDEF For reaction conditions when the only effect on the environment is the emission or absorption of heat: the second law of thermodynamics takes the form: Gibbs’ free energy (not a true form of energy, contains an entropy term) 2011.09.13.. TÁMOP –4.1.2-08/2/A/KMR-2009-0006 13 Biochemistry: Thermodynamics in metabolism Relation between free energy and equilibrium 1. free energy change free energy change for standard conditions (1 M [reactant], pH 7.0, 25°C) gas constant 2011.09.13.. TÁMOP –4.1.2-08/2/A/KMR-2009-0006 14 Biochemistry: Thermodynamics in metabolism At equilibrium Relation between free energy and equilibrium 2. 2011.09.13.. TÁMOP –4.1.2-08/2/A/KMR-2009-0006 15 Biochemistry: Thermodynamics in metabolism Relation between free energy and equilibrium 3. Mass action ratio 2011.09.13.. TÁMOP –4.1.2-08/2/A/KMR-2009-0006 16 Biochemistry: Thermodynamics in metabolism Interpretation of free energy changes in metabolism Reaction .G0kJ/mol .G kJ/mol Type hexokinase -20.9 -27.9 non-equilibrium G6P isomerase +2.1 -1.3 near-equilibrium PFK -17.1 -26.5 non-equilibrium aldolase +23.0 -6.1 near-equilibrium GA3PDH+PGAkinase +7.9 -11.5 near-equilibrium phosphoglyceromutase +4.6 -0.5 near-equilibrium enolase -3.3 -2.4 near-equilibrium pyruvatekinase -24.7 -15.8 non-equilibrium Thermodynamic characteristics of the glycolyticreactions in rat heart 2011.09.13.. TÁMOP –4.1.2-08/2/A/KMR-2009-0006 17 Biochemistry: Thermodynamics in metabolism Relation between kinetics and thermodynamics 1. At equilibrium 2011.09.13.. TÁMOP –4.1.2-08/2/A/KMR-2009-0006 18 Relation between kinetics and thermodynamics 2. Biochemistry: Thermodynamics in metabolism 2011.09.13.. TÁMOP –4.1.2-08/2/A/KMR-2009-0006 19 Biochemistry: Thermodynamics in metabolism Relation between kinetics and thermodynamics 3. For a metabolic pathway in steady state: Kinetic interpretation of near-and non-equilibrium reactions: •near-equilibrium: • non-equilibrium: 2011.09.13.. TÁMOP –4.1.2-08/2/A/KMR-2009-0006 20 Biochemistry: Thermodynamics in metabolism Coupling-in-series in metabolismDEF Reaction .G0kJ/mol .G kJ/mol Type hexokinase -20.9 -27.9 non-equilibrium G6P isomerase +2.1 -1.3 near-equilibrium PFK -17.1 -26.5 non-equilibrium 2011.09.13.. TÁMOP –4.1.2-08/2/A/KMR-2009-0006 21 Biochemistry: Thermodynamics in metabolism Coupling-in-parallel in metabolismDEF1. M L B A X Y X Y ATP ADP NAD NADH2 NADP NADPH2 coenzymeA acetyl coenzymeA 2011.09.13.. TÁMOP –4.1.2-08/2/A/KMR-2009-0006 22 Biochemistry: Thermodynamics in metabolism Coupling-in-parallel in metabolism 2. glucose-6-phosphate glucose Pi H2O .G0=+13.8 kJ/mol .G<0:[glucose]>1,6 M (~3.000 ×[glucose]norm) ADP ATP H2O Pi .G0=-30.5kJ/mol 2011.09.13.. TÁMOP –4.1.2-08/2/A/KMR-2009-0006 23 Biochemistry: Thermodynamics in metabolism Coupling-in-parallel in metabolism 3. glucose-6-phosphate glucose ATP ADP .G0=-16.7kJ/mol 2011.09.13.. TÁMOP –4.1.2-08/2/A/KMR-2009-0006 24 Biochemistry: Thermodynamics in metabolism Kinetic and thermodynamic structure of metabolic pathways Biological significance: • non-equilibrium reactions (directionality, regulation) • near-equilibrium reactions (reversibility) 2011.09.13.. TÁMOP –4.1.2-08/2/A/KMR-2009-0006 25 Biochemistry: Enzyme kinetics in metabolism Relation between kinetics and thermodynamics •thermodynamics: spontaneity and direction of the process • kinetics: rate of the process activation energyDEF 2011.09.13.. TÁMOP –4.1.2-08/2/A/KMR-2009-0006 26 Biochemistry: Enzyme kinetics in metabolism Factors decreasing the activation energy during enzyme action •proximity of substrates • spatial orientation • forced positional strain • additional interactions with functional groups 2011.09.13.. TÁMOP –4.1.2-08/2/A/KMR-2009-0006 27 Biochemistry: Enzyme kinetics in metabolism Structure of enzymes 1: Amino acids 2011.09.13.. TÁMOP –4.1.2-08/2/A/KMR-2009-0006 28 Biochemistry: Enzyme kinetics in metabolism Common amino acids 2011.09.13.. TÁMOP –4.1.2-08/2/A/KMR-2009-0006 29 Biochemistry: Enzyme kinetics in metabolism Structure of enzymes 2: Peptide bondsDEF Primary structure 2011.09.13.. TÁMOP –4.1.2-08/2/A/KMR-2009-0006 30 Biochemistry: Enzyme kinetics in metabolism Structure of enzymes 3: Higher levels of protein organization .-helix ß-sheet Secondary structureDEF 2011.09.13.. TÁMOP –4.1.2-08/2/A/KMR-2009-0006 31 Biochemistry: Enzyme kinetics in metabolism Structure of enzymes 4: Higher levels of protein organization Tertiary structureDEF 2011.09.13.. TÁMOP –4.1.2-08/2/A/KMR-2009-0006 32 Enzymes in action (changes in conformationDEF) Biochemistry: Enzyme kinetics in metabolism hexokinase glucose Peptid hasitas.jpg substrate transient state products Biochemistry: Enzyme kinetics in metabolism Hydrolysis of peptide bonds (general mechanism) 2011.09.13.. TÁMOP –4.1.2-08/2/A/KMR-2009-0006 33 Biochemistry: Enzyme kinetics in metabolism Catalytic mechanism of serine proteasesDEF proteases catalyze the hydrolysis of peptide bonds in proteins Energy stages in the course of reaction 2011.09.13.. TÁMOP –4.1.2-08/2/A/KMR-2009-0006 34 Biochemistry: Enzyme kinetics in metabolism 2011.09.13.. TÁMOP –4.1.2-08/2/A/KMR-2009-0006 35 A closer look at the active siteDEFof chymotrypsin Biochemistry: Enzyme kinetics in metabolism 2011.09.13.. TÁMOP –4.1.2-08/2/A/KMR-2009-0006 36 Role of the separate amino acids in the active site -the hydroxyl oxygen of Ser195 loses its hydrogen to His57 -the nucleophilicoxygen attacks the carbonyl C of the peptide bond Biochemistry: Enzyme kinetics in metabolism 2011.09.13.. TÁMOP –4.1.2-08/2/A/KMR-2009-0006 37 First transient tetrahedral state of the enzyme-substrate complex The tetrahedral structure of the transient enzyme-substrate complex is stabilized by two amide hydrogens coordinating the anionic oxygen in the oxyanionhole. Energy stages in the course of reaction Biochemistry: Enzyme kinetics in metabolism 2011.09.13.. TÁMOP –4.1.2-08/2/A/KMR-2009-0006 38 The tetrahedral structure decomposes to an acyl-enzyme intermediate and a peptide with new N-terminal portion is released. Cleavage of the peptide bond Energy stages in the course of the reaction Biochemistry: Enzyme kinetics in metabolism 2011.09.13.. TÁMOP –4.1.2-08/2/A/KMR-2009-0006 39 Second transient tetrahedral state of the enzyme-substrate complex -a water molecule enters and attacks the ester bond of the enzyme-substrate complex -a second tetrahedral structure is formed as one of the waters hydrogensis passed to His57 Biochemistry: Enzyme kinetics in metabolism 2011.09.13.. TÁMOP –4.1.2-08/2/A/KMR-2009-0006 40 Cleavage of the ester bond linking the enzyme and the substrate The tetrahedral structure decomposes releasing a product peptide with new C-terminal portion. Ser195 recovers its hydrogen from His57 and the initial state of the enzyme is restored. Energy stages in the course of the reaction 2011.09.13.. TÁMOP –4.1.2-08/2/A/KMR-2009-0006 41 Biochemistry: Enzyme kinetics in metabolism Mechanism of action of protease inhibitors Most natural inhibitors are proteins which act as pseudosubstratesblocking the catalytic mechanism at different stages 1. Reversible inhibitors (e.g. pancreatic trypsininhibitor): bind, but the first tetrahedral structure is not formed protease inhibitor pseudosubstrateloop 2011.09.13.. TÁMOP –4.1.2-08/2/A/KMR-2009-0006 42 Biochemistry: Enzyme kinetics in metabolism Mechanism of action of protease inhibitors 2. 2. Irreversible inhibitors (e.g. serpinsDEF): the first tetrahedral structure (IPM) and the acylatedenzyme-substrate complex (IPacyl) are formed, but the ester bond cannot be cleaved, because the catalytic site is distorted 2011.09.13.. TÁMOP –4.1.2-08/2/A/KMR-2009-0006 43 Messages to take home 1) Biochemistry studies the chemical reactions in living organisms 2) The reductionist approach dissects metabolism to separate reactions and builds up the whole system from these pieces of knowledge 3) Thermodynamics determines the directionality of metabolic pathways and its principles are valid for each separate reaction as well as for sequences of reactions 4) Spontaneity of reactions under the concentration, temperature and pressure conditions of living organisms is maintained by their coupling-in-series and coupling-in-parallel 5) Near-equilibrium reactions have free energy change approaching zero, whereas non-equilibrium reactions have large negative free energy change 6) Proteins are composed of amino acids bound through peptide bonds and polypeptide chains form higher-order spatial structures 7) Specific interactions between amino acid residues in the active site of enzymes and the substrates result in a decrease in the activation energy of the catalyzed reaction (e.g. serine protease) Biochemistry: Enzyme kinetics in metabolism 2011.09.13.. TÁMOP –4.1.2-08/2/A/KMR-2009-0006 44 Biochemistry: Enzyme kinetics in metabolism Comprehension problem 1 Which statement is true regarding the direction of the biochemical processes? A. Spontaneous reactions are accompanied by positive changes in the free enthalpy. B. Spontaneous reactions are accompanied by positive changes in the free entropy. C. Spontaneous reactions are accompanied by positive changes in the free energy. D. Spontaneous reactions are accompanied by negative changes in the free enthalpy. E. Spontaneous reactions are accompanied by negative changes in the free energy. 2011.09.13.. TÁMOP –4.1.2-08/2/A/KMR-2009-0006 45 Biochemistry: Enzyme kinetics in metabolism Comprehension problem 2 What is the maximal efficiency at which the energy released from glucose oxidation is converted into energy that can be used as ATP? A. 90 % B. 75 % C. 50 % D. 30 % E. 10 % 2011.09.13.. TÁMOP –4.1.2-08/2/A/KMR-2009-0006 46 Biochemistry: Enzyme kinetics in metabolism Comprehension problem 3 What is the relation between equilibrium and the change in the free energy of a reaction? A. At equilibrium the free energy is not changed. B. At equilibrium the free energy change is equal to the standard free energy change. C.At equilibrium the free energy change is always positive. D.At equilibrium the free energy change is always negative. E. The term free energy change can be applied only for reactions in equilibrium. 2011.09.13.. TÁMOP –4.1.2-08/2/A/KMR-2009-0006 47 Biochemistry: Enzyme kinetics in metabolism Comprehension problem 4 Which statement is true regarding reactions coupled in parallel in metabolic pathways? A. The presence of cofactor is obligatory. B. The presence of coenzyme is obligatory. C. The presence of prosthetic group is obligatory. D. The reactions are accompanied always by negative standard free energy change. E. The sign of the change in the free energy is the same for the coupled reactions (a reaction with positive change in the free energy cannot be coupled-in-parallel to a reaction with negative change). 2011.09.13.. TÁMOP –4.1.2-08/2/A/KMR-2009-0006 48 Biochemistry: Enzyme kinetics in metabolism Comprehension problem 5 Which statements are true regarding non-equilibrium reactions in metabolic pathways? 1. They determine the direction of the pathway. 2. They are targets of regulation. 3. They are reversible. 4. They confer reliability of the pathway. 5. They can participate in multiple pathways. A. 1,2B. 3,4C. 4,5D. 3,5E. 2,4 2011.09.13.. TÁMOP –4.1.2-08/2/A/KMR-2009-0006 49 Biochemistry: Enzyme kinetics in metabolism Comprehension problem 6 Which statement is true regarding the role of enzymes in metabolic pathways? A. They determine the direction of the reaction. B. They determine the rate of the reaction. C. They determine the free energy change of the reaction. D. They determine the enthalpy change of the reaction. E. None of the above 2011.09.13.. TÁMOP –4.1.2-08/2/A/KMR-2009-0006 50 Biochemistry: Enzyme kinetics in metabolism Comprehension problem 7 At body temperature proteins are stable molecules, because A. their synthesis is accompanied by large negative free energy change. B. their degradation is accompanied by large negative free energy change. C. their hydrolysis requires high activation energy. D. their hydrolysis requires no or only minimal activation energy. E. their hydrolysis occurs only at significantly higher temperatures. 2011.09.13.. TÁMOP –4.1.2-08/2/A/KMR-2009-0006 51 Biochemistry: Enzyme kinetics in metabolism Comprehension problem 8 Select the functional group which performs the nucleophilicattack in the course of the catalytic action of serine proteases. A. carboxyl. B. hydroxyl. C. imidazole. D. sulfhydryl. E. none of the above. 2011.09.13.. TÁMOP –4.1.2-08/2/A/KMR-2009-0006 52 Biochemistry: Enzyme kinetics in metabolism Comprehension problem 9 Select the type of bond transiently formed between the substrate and the enzyme in the course of the catalytic action of serine proteases. A. ether. B. ester. C. peptide. D. amide. E. acidic anhydrate. 2011.09.13.. TÁMOP –4.1.2-08/2/A/KMR-2009-0006 53 Biochemistry: Enzyme kinetics in metabolism Comprehension problem 10 Which statement is true regarding the function of protease inhibitors? A. Only the structure of the enzyme is changed during the inhibition of the protease. B. Only the structure of the inhibitor is changed during the inhibition of the protease C. The structure of both enzyme and inhibitor is changed during the inhibition of the protease. D. Following the inhibition of the protease active inhibitor can always be released from the complex. E. The inhibition of the protease is always reversible. 2011.09.13.. TÁMOP –4.1.2-08/2/A/KMR-2009-0006 54 Recommended literature Orvosi Biokémia (Ed. Ádám Veronika): pp. 21-30, 55-60 medicina_hu-891.jpg Biochemistry: Enzyme kinetics in metabolism 2011.09.13.. TÁMOP –4.1.2-08/2/A/KMR-2009-0006 55 Biochemistry: Enzyme kinetics in metabolism Answers to comprehension problems 1. E; 2. D; 3. A; 4. A; 5. A; 6. B; 7. C; 8. B; 9. B; 10. C