BMB Lecture 1 September 27, 2017

Transcription

1 BMB Lecture 1 September 27, 2017 Introduction to Enzyme Catalysis Transition State Theory

2 Welcome to BMB/Ch 178 Macromolecular function: Kinetics and Mechanisms Wednesdays and Fridays 10:30 am 12:00 pm 101 Schlinger Lecturer: Shu-ou Shan x3879, TAs: Chien-I Yang, Jae Ho Lee, Hao Hsuan Hsieh

3 Enzymology: Past, Present and Future

4 Enzymology: Past, Present and Future Enzyme design/engineering

5 Goals Develop a sense of the energetic principles and molecular mechanisms that underlie biological function; Be able to formalize ideas / models in molecular & energetic terms and subject them to rigorous tests. Understand what molecular process is being measured under an experimental setup, and vice versa; Develop the ability to translate rate and equilibrium information into a model of molecular actions.

6 Example: MP selection at the membrane Rao et al, submitted

7 Example: MP selection at the membrane Rao et al, submitted

8 Example: MP selection at the membrane Rao et al, submitted

9 Example: MP selection at the membrane Rao et al, submitted

10 BMB/Ch 178 Syllabus 9/27 Introduction; review of basic thermodynamics; transition state theory 9/29 Transition state theory and ramifications; pymol tutorial PS1 posted 10/4 Covalent catalysis; General acid/base catalysis; 10/6 metal ions; electrostatics and hydrogen bonds 10/11 Entropy, intrinsic binding energy and the circe effect 10/13 Specificity and editing; promiscuity and enzyme evolution PS1 due 10/18 Cooperativity and allostery PS2 posted 10/20 Kinetics 101: rate laws, rate-limiting steps 10/25 Steady state kinetics (I) 10/27 Steady state kinetics (II); Presteady-state kinetics (I)

11 BMB/Ch 178 Syllabus (continued) 11/1 Literature Discussion (I) 11/3 Literature Discussion (II) PS2 due 11/8 Pre-steady state kinetics (II) PS3 posted 11/10 Pre-steady state kinetics (III); overview of analytical tools 11/15 Kinetic simulations 11/17 Single Molecule kinetics (I) 11/22 Single Molecule kinetics (II) 11/24 No class (Thanksgiving) 11/29 Enzyme design and evolution; PS3 due 12/1 Literature Discussion (III) TQFR 12/4 Final posted 12/8 Finals due by 6PM. In front of Shu-ou s office (109 Braun).

12 A Few Practical Notes Take time to let concepts sink in Learn how we arrived at the conclusions, rather than remembering the conclusions Read recommended literature (available on course website) Ask questions!

13 Textbooks - Fersht, Structure and Mechanisms in Protein Science, Freeman, 3rd ed., (on reserve in the Library) - Jencks, Catalysis in Chemistry and Enzymology, Dover, 1987

14 Softwares Pymol: structure viewing download from: Berkeley Madonna: kinetic simulations download beta version from:

15 Rates of most biological reactions without enzyme

16 Magnitute of Enzyme Catalysis Enzyme k cat / k uncat Sweet potato β-amylase Orotidine decarboxylase Fumarase Mandelate racemase Staphylococcal nuclease > Carboxypeptidase B AMP nucleosidase Adenosine deaminase Ascites tumor dipeptidase Cytidine deaminase Ketosteroid isomerase Phosphotriesterase Triosephosphate isomerase Carbonic Anhydrase Chorismate mutase Catalytic antibodies Chemical catalysts

17 Why Are Enzymes so Efficient? A thought experiment from hypothetical enzyme models

18 Hypothetical Enzymatic reaction

19 Hypothetical Enzyme 1: Catalytic residues

20 Hypothetical Enzyme 2: Positioned Catalytic residues

21 Hypothetical Enzyme 3: Positioned Binding and Catalytic residues Binding interactions localize substrate to the active site But substrate is not positioned properly

22 Energetic costs of localizing a substrate to enzyme active sites Loss of translational and rotational entropy Cost for desolvation Redundant interactions are required!

23 Hypothetical Enzyme 3b: Positioned Binding and Catalytic residues Binding and Catalytic interactions are interconnected - optimal catalysis requires positioning

24 Hypothetical Enzyme 4: Tuning Interactions and Binding Energy 1. Multiple H bonds compensate for desolvation from bulk water 2. Microelectrostatic environment accentuates catalytic potential

25 Problem: too strong binding slows down product dissociation and decreases specificity

26 Hypothetical Enzyme 4: Tuning Interactions and Binding Energy Conformational change allows substrate ingress and product egress

27 Hypothetical Enzyme 4: Tuning Interactions and Binding Energy Ground state destabilization: maximize the contribution of catalytic interactions. Increases specificity and speeds turnover.

28 A brief review of thermodynamics Deals with energies of chemical processes without reference to the specific pathway or molecular properties Divide the universe arbitrarily into System + Surrounding Examine the energy change of a given process: E = E final - E i Most biochemistry is performed under constant pressure; Convenient to define H = E + PV ~ E (for processes in solution)

29 Let s examine a simple biochemical reaction H Measure H -> Large and negative Duplex DNA forms because the enthalpy of system (DNA) decreases. 1 st Law of Thermodynamics: energy of universe stays constant, i.e., E universe = 0 What determines the direction of a spontaneous change in nature???

30 Entropy S = Entropy = k B lnw W = statistical weight = N! n 1!n 2!n 3!n 4!... k B = Boltzman constant 2 nd Law of Thermodynamics: entropy of the universe increases for a spontaneous process Our ruler for spontaneous changes is entropy change of the universe, but as scientists, we also would like to focus on the system

31 Gibbs Free Energy G = H T S for constant press. & temp. processes A way to look at the total entropy change of system + surrounding, while focusing on the system Predict direction of spontaneous changes: G > 0 unfavorable process G < 0 favorable process Directly connect to chemical equilibria measurements: G = RT lnk

32 Exercise: What will happen? A + B C + D G (kj / mol)

33 Transition State Theory K S S ν P K = [S ]/[S] d[p]/dt = ν[s ] d[p]/dt = ν [S] K = (k B T/h) [S] exp (- G /RT) k = (k B T/h) exp (- G /RT) Relates reaction rate to the difference in free energy between ground state and transition state Allows equilibrium analysis to be applied to reaction rates by treating transition state as a chemical species in pseudo equilibrium with substrate

34 Substrates fit enzymes as a key fits a lock. Emil Fischer?

35 Catalysts must bind transition state much stronger than substrate I believe that an enzyme has a structure closely similar to that found for antibodies, but with one important difference, namely, that the surface configuration of the enzyme is not so closely complementary to its substrate but is instead complementary to an unstable molecule with only transient existence namely, the activated complex for the reaction that is catalyzed by the enzyme. The assumption made above that the enzyme has a configuration complementary to the activated complex, and accordingly has the strongest power of attraction for the activated complex, means that the activation energy for the reaction is less in the presence of the enzyme than in its absence, and accordingly that the reaction would be speeded up by the enzyme. Linus Pauling,1945

36 Transition State Theory and Enzymatic Catalysis Lienhard, Science 180: 149 (1973)

37 Quiz 1. Must an an enzyme bind S more tightly than S? 2. What does the theory say about the path to the transition state? 3. Must the enzyme be a pre-existing template for the transition state?

38 Role of Conformational Changes in Enzyme Catalysis 1. Enzyme structure tends to change with inhibitor binding 2. Most transition state inhibitors exhibits slow binding

39 Quiz 1. Must an an enzyme bind S more tightly than S? 2. What does the theory say about the path to the transition state? 3. Must the enzyme be a pre-existing template for the transition state? 4. What if the mechanisms of spontaneous and enzyme-catalyzed reactions are different?

40 An observed reaction goes through the lowest energy transition state

41 Quiz 1. Must an an enzyme bind S more tightly than S? 2. What does the theory say about the path to the transition state? 3. Must the enzyme be a pre-existing template for the transition state? 4. What if the mechanisms of spontaneous and enzyme-catalyzed reaction differ? 5. Is transition state theory adaptable to multi-substrate reactions? 6. Is transition state theory adaptable to enzymatic reactions that go through tunneling?

42 Does the transition state affinity depend on a few or many interactions? Intrinsic binding energy >> apparent transition state stabilization Energetics at enzyme active sites are NOT additive Enzymes exploit multiple interactions to the fullest extent

43 Test of Transition State Theory: Transition State Analogue Inhibitors Cytidine deaminase: fold rate enhancement

44 Application of Transition state Theory: Catalytic Antibodies Example 1: Lactone Cyclization Napper et al, Science 237, 1041 (1987)

45 Example 2: Diels-Alder Reaction S ~ -30 to -40 e.u. Diene 1 (mm) Km Dienophile 2 (mm) k cat (s -1 ) k 2 (uncat) (M -1 s -1 ) K i (nm) k cat / k 2 (mm) K m 1 K m 2 / K i (mm) Braisted & Schultz, JACS 112: 7430 (1990)

46 Recommended Readings CHALLENGES IN ENZYME MECHANISM AND ENERGETICS Daniel A. Kraut, Kate S. Carroll, and Daniel Herschlag Annu. Rev. Biochem : Enzymatic catalysis and transition state theory Gustav E. Lienhard, Science 180, p149 (1973)