PETER PAZMANY CATHOLIC UNIVERSITY Consortium members SEMMELWEIS UNIVERSITY, DIALOG CAMPUS PUBLISHER

Transcription

1 PETER PAZMANY SEMMELWEIS CATHOLIC UNIVERSITY UNIVERSITY 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. 1

2 Peter Pazmany Catholic University Faculty of Information Technology INTRODUCTION TO BIOPHYSICS (Bevezetés a biofizikába) ENZYMES (Enzimek) GYÖRFFY DÁNIEL, ZÁVODSZKY PÉTER 2

3 Introduction Catalysts are substances that can accelerate reactions taking place spontaneously even in the absence of the catalyst Catalysts are reclaimed in unchanged form after the reaction occurs Enzymes are catalysts of biological processes Enzymes are proteins often containing cofactors such as metal ions 3

4 The substance converted in the reaction catalyzed by the enzyme is called the substrate The substance produced in this reaction is called product 4

5 Classification of enzymes Enzymes are classified into six groups based on the type of reaction catalyzed by them The coenzyme is responsible for the type of reaction to be catalyzed while the apoenzyme determines the type of the substrate converted in the reaction 5

6 Classes of enzymes Class Catalyzed reaction Example Oxidoreductases Oxidation-reduction GAPDH Transferases Group transfer ERK2 Hydrolases Hydrolysis Trypsin Lyases Double bond formation or removal Fumarase Isomerases Isomerization Triose phosphate isomerase Ligases Ligation DNA ligase 6

7 GAPDH 7

8 MAP kinase ERK2 8

9 Trypsin 9

10 Fumarase 10

11 Triose phosphate isomerase 11

12 DNA ligase 12

13 Enzymes in action Enzymes can catalyze only reactions taking place spontaneously, i.e. in the absence of catalyst Reactions can take place spontaneously if the free energy of products is less than that of the reactants Thus, catalysts such as enzymes do not affect the thermodynamics of reactions but influence their kinetics 13

14 Usually, for a reaction to take place, an energy barrier must be passed The height of this barrier will determine the rate of the reaction The higher the barrier the slower the reaction Catalysts can make this barrier lower, and thus accelerate the reaction even by several orders of magnitude Now let us examine where this barrier comes from 14

15 Recall: Arrhenius theory The Swedish chemist Arrhenius found a relation between the rate of reaction and the temperature The Arrhenius equation says: k =A e E a / R T where k is the rate constant, Ea is the activation energy, R is the gas constant, T is the temperature and A is a constant called Arrhenius constant or pre-exponential factor 15

16 k as a function of T 16

17 Recall: Boltzmann distribution Ludwig Boltzmann found the energy distribution of particles in a system at equilibrium The Boltzmann distribution is: 1 E /k p E i = e Z i B T where p(ei) is the probability that a particle is in a state having Ei energy, kb is the Boltzmann constant and Z is the partition function 17

18 The partition function Ei / k B T Z = e i is the sum of the Boltzmann factors for all of the i states and serves as a scaling factor to ensure that the sum of probabilities equals 1 18

19 Boltzmann distribution 19

20 Thus, the rate of a reaction is proportional to the fraction of particles having an energy higher than the activation energy Hence, if a catalyst lowers the activation energy, a higher fraction of particles will have an energy higher than the activation energy thus, the rate of the reaction will be higher 20

21 Fraction of particles of energy E in an uncatalyzed and a catalyzed reaction 21

22 Recall: reaction profile The progress of a reaction can be characterized by one or more reaction coordinates Now, let us consider a reaction with one reaction coordinate The free energy of the system can be plotted as a function of a reaction coordinate This plot is called reaction profile 22

23 Reaction profile 23

24 The effect of a catalyst on the reaction profile is that it lowers the activation energy and thus lowers the barrier that must be passed for the system the reaction to occur It can be seen that a catalyst accelerates a reaction not only in one direction but in the opposite direction as well However, enzymes do not alter the reaction free energy Gr, and therefore do not influence whether the reaction occurs spontaneously 24

25 Effect of a catalyst on the reaction profile 25

26 Hypotheses for enzyme action Several hypotheses have been proposed to explain how enzymes can accelerate reactions even by several orders of magnitude Enzymes often open up a by-pass pathway with lower activation energy for the reaction, which can thus proceed faster 26

27 Transition state stabilization Let us consider the reaction to be catalyzed by an enzyme as K k S S P where S is the substrate, S is the transition state, P is the product, K is the equilibrium constant for the formation of the transition state from the substrate, and k is the rate constant of the conversion of the transition state to the product 27

28 It is assumed that the equilibrium step of the reaction is far faster than the second step Thus, the overall rate of the reaction can be approximated by v =k [S ] k [S ] It can be seen that the rate of the overall reaction is proportional to the concentration of the transition state 28

29 Since [S ] G / RT K = =e [S ] where ΔG is the activation free energy, describing the stability of transition state relative to the substrate The more stable the transition state the higher its concentration Thus, enzymes accelerate reactions by stabilizing the transition state 29

30 Enzyme-substrate complex In the course of catalysis, a complex of the enzyme and the substrate(s) is formed The transition state is also formed in an enzyme substrate complex The specificity of enzymes is brought about by the specific binding of substrate The region of the enzyme where the binding occurs is called the active site 30

31 Lock and key hypothesis To explain substrate specificity, several theories have been proposed The lock and key hypothesis assumes that the shape of the active site is a negative of the shape of the substrate Later, several enzymes were found to be able to catalyze the reaction of substrates having significantly different shapes but not of substances having almost the same shape as a known substrate 31

32 The lock and key hypothesis 32

33 Induced fit hypothesis Due to the above mentioned difficulties, a new model has been proposed to better explain substrate specificity This new model, called induced fit model assumes that, when the substrate approaches the active site of the enzyme, a conformational change occurs in the enzyme, allowing the binding based on shape complementarity 33

34 The induced fit mechanism 34

35 Transition state fit According to a more modern view, it is the transition state whose shape fits the shape of the active site Thus, a lock and key binding occurs not between the enzyme and the ground state but between the enzyme and the transition state of the substrate 35

36 Transition state fit 36

37 The transition state is a high-energy state of the substrate According to the Boltzmann distribution, states with high energy have a low but non-zero probability to occur Thus, a small amount of substrate molecules in the transition state is present in the medium 37

38 Transition state fit occurs when the enzyme selects a substrate molecule being in the transition state for binding rather than molecules in the ground state Not only the enzyme can select from the reservoir of substrate states but substrates can also select from the preexisting enzyme conformations This mechanism is called conformational selection or fluctuation fit 38

39 Conformational selection by the enzyme 39

40 Conformational selection by the substrate 40

41 Transition-state analogues Based on the model assuming the selective binding of the transition state by the enzyme, it has been proposed that analogues of the transition state compounds, that is a compound having similar conformation to it should be good inhibitors of the enzyme Indeed, several observations have been accumulated that support the concept that transition state analogues are good inhibitors 41

42 Abzymes The existence of abzymes lends further support for the transition state fit model Abzymes are catalytic antibodies They have catalytic activity for reactions for which they can selectively bind the transition state Immunizing animals by a transition state analogue, an effective enzyme can be obtained 42

43 Michaelis-Menten model for enzyme kinetics The Michaelis-Menten model of enzyme kinetics accounts for dependence of the rate of the enzyme reactions on the substrate concentration A steady-state approximation has been used to construct a model fitting well the experimental results for many enzymes 43

44 Kinetic curves of an enzyme reaction 44

45 The following scheme can be proposed for a generic enzyme reaction k1 k2 k 1 k 2 E S ES P where E is the enzyme and S is the substrate in their free forms, ES is the enzyme-substrate complex and P is the product The corresponding rate constants are also shown 45

46 Assuming that the rate of formation of product from the enzyme-substrate complex is far higher than the rate of the reverse reaction, that is k 2 k 2 the general scheme of enzyme reactions can be simplified to be k1 k2 E S ES P k 1 46

47 The rate of the reaction is assumed to be v 0=k 2 [ ES ] Since we do not know the concentration of the enzyme-substrate complex, we need to express it in terms of the known quantities such as the initial substrate or enzyme concentration d [ ES ] =k 1 [ E ][S ] k 1 k 2 [ ES ] dt 47

48 Making use of the steady state approximation, i.e. that the concentration of the enzymesubstrate complex does not change for a wide time range d [ ES ] =0 dt and thus k 1 [ E ][S ]= k 1 k 2 [ ES ] 48

49 After rearrangement we obtain [ E ][ S ]/[ ES ]= k 1 k 2 / k 1 If we define a new constant called Michaelis constant, KM K M = k 1 k 2 / k 1 we get a simpler equation [ E ][S ] [ ES ]= KM 49

50 The concentration of the free enzyme can be obtained from the equation [ E ]=[ E ] T [ ES ] where [E]T is the total enzyme concentration Since the total amount of the enzyme does not change through the reaction, it will be equal to the amount of enzyme initially put into the reaction mixture 50

51 Substituting the expression for the enzyme concentration into the equation above, we get [ ES ]= [E ]T [ ES ] [S ] KM Solving the equation for [ES], we obtain [S] [ ES ]=[ E ]T [S ] K M 51

52 Substituting this expression into the equation for the reaction rate, we obtain [S ] v 0=k 2 [ E ]T [S ] K M The reaction can proceed with the maximal speed when all of the enzyme molecules are in complex with a substrate molecule, that is when [ ES ]=[ E ]T 52

53 Thus the maximal velocity is v max =k 2 [ E ]T Based on this, the relationship between the maximal and the actual velocity is [S] v 0=v max [ S ] K M 53

54 It can be seen in the equation above that KM corresponds to the substrate concentration where the rate of reaction is half of the maximal rate It also shows that the Michaelis constant is an important kinetic property of enzymes 54

55 The rate of the reaction as a function of the substrate concentration 55

56 If the substrate concentration is far lower than the Michaelis constant, that is [S ] K M then the rate of reaction is approximately v max v 0 [S] KM It can be seen that at low substrate concentration, the reaction is first-order with respect to the substrate 56

57 On the other hand, if the substrate concentration is far higher than the Michaelis constant, that is [S ] K M then the rate of reaction is approximately v 0 v max It can be seen that at high substrate concentration, the reaction is zeroth-order with respect to the substrate 57

58 Catalytic efficiency The turnover number of an enzyme is the number of molecules converted into a product in unit time when the enzyme is fully saturated by the substrate The turnover number is equal to the rate constant k2 which is also called kcat The maximal velocity, vmax in terms of kcat is v max =k cat [ E ] T 58

59 When the substrate concentration is far lower than the Michaelis constant, the enzymatic rate is much less than kcat From equation v 0=k cat [ ES ] and [ E ][S ] [ ES ]= KM we can obtain a new equation 59

60 k cat v 0= [ E ][ S ] KM kcat/km behaves as a second-order rate constant for the reaction between the substrate and the free enzyme, and thus can serve as a measure of catalytic efficiency The physical limit on the value of kcat/km is the rate constant of formation of the enzymesubstrate complex which cannot be faster than allowed by the velocity of diffusion 60

61 Inhibitors Enzymes can be inhibited by specific inhibitors Two main classes of inhibitors can be distinguished Competitive inhibitors Non-competitive inhibitors Competitive inhibitors use the same binding site on the enzyme as the substrate and a competition occurs between the substrate and the inhibitor for the binding site 61

62 Non-competitive inhibitors bind to a different site on the enzyme than the substrate They cause a conformational change in the enzyme, leading to a reduction of the action of the enzyme Competitive and non-competitive inhibitors have a different effect on the kinetics of the enzyme reaction and thus they can be kinetically distinguished 62

63 In the case of a competitive inhibitor, if the concentration of substrate is high enough, the maximal velocity, vmax, can be attained but the substrate concentration where the velocity is the half of vmax, KM, will be higher In the case of non-competitive inhibitors, the maximum velocity vmax cannot be attained even at very high substrate concentration, but the substrate concentration where the velocity is the half of the modified maximal velocity is unchanged 63

64 Competitive inhibitor 64

65 Non competitive inhibitor 65

66 Catalytic strategies The function of enzymes is based on one or more of a few strategies Through covalent catalysis, a reactive group of the active site becomes covalently modified In the active site of trypsin, the catalytic serine residue forms an acyl-enzyme intermediate with the N-terminal part of the cleaved polypeptide chain 66

67 Acyl-enzyme intermediate in the active site of trypsin 67

68 In acid-base catalysis, a proton transfer occurs where the donor or acceptor group is not water Metal ions can take part in the catalytic reactions in several ways, for example they can supply positive charge if the intermediate is negatively charged, or they can take part in the substrate binding The enzyme can help substrates to approach each other in a proper orientation, entropically decreasing the activation free energy 68

69 Ribozymes Catalytic capability is a property not exclusively of proteins but also of RNAs Several catalytic RNAs called ribozymes are known Ribozymes take part mainly in the catalysis of reactions related to RNA conversion Ribozymes are important constituents of ribosomes, the molecular machines responsible for protein synthesis 69

70 Large subunit of a bacterial ribosome 70

71 Small subunit of a bacterial ribosome 71

72 Another important process catalyzed partly by ribozymes is splicing through which exons are cleaved out from the premature mrna molecule Inspired by the discovery of catalytic RNAs, an evolutionary concept called the RNA world was proposed According to these hypothesis, at an earlier stage of evolution, it was RNA that was responsible for catalysis and information storage instead of proteins and DNA, respectively 72

73 RNA splicing 73

74 The RNA world hypothesis is supported by the existence of catalytic RNAs and the fact that many enzymes have a coenzyme, i.e. a ribonucleotide derivative such as NAD, the most important electron carrier molecule of the cell and ATP, the most important energy currency 74

75 Adenosine triphosphate (ATP) 75

76 Nicotinamid adenine dinucleotide (NAD) 76