1. Energy and life 1 st law of thermodynamics: Law of Conservation of Energy. Energy cannot be created or destroyed Then why do we talk about the “energy crisis?” What does it mean to be phototrophic vs chemotrophic? (Light as energy source vs chemical energy source) What does ATP synthetase or photosynthetic reaction center do? Chapter 8: Energy, enzymes, and regulation

2. Energy transduction Enzymes can convert one form of energy into another form. * Examples? Myosin in muscle: ATP synthase: Flagellum: Photosynthetic reaction center: Electron transfer chain in mitochondria: chemical to mechanical energy transmembrane gradient into chemical energy transmembrane gradient into motion light into transmembrane gradient chemical energy into transmembrane proton gradient

3. 2 nd law of thermodynamics: entropy (disorder) of an isolated system always increases Is a living organism in a relatively low or high state? How to grow from a seed or an embryo to an adult organism? Decrease in entropy? Entropy: A measure of the randomness or disorder of a system The greater the disorder the greater the entropy

4. Energy = The capacity to do work or to cause particular changes. Chemical work The synthesis of complex biological molecules from simpler precursors Mechanical work Changing the location of organisms (e.g., flagellum), cells and structures within cells Transport work The ability to transport molecules against a concentration gradient (uptake of nutrients, elimination of waste, maintenance of ion balance)

5. Efficiency of energy conversion? Less than 100%. Question: Where does the rest go? Heat: thermal motion of molecules without (strong) thermal gradient. It is often difficult to capture this form of energy for doing work Idea: Eventual thermal death of the universe. Is being debated. Bottom line for biology: living systems need input of energy to keep functioning. Question: what is the overall energy source driving the biosphere on earth? Sun light: photosynthesis

6.  G =  H - T  S  G = change in free energy (amount of energy available to do work) Describes direction of spontaneous processes. Reactions with a negative  G value will occur spontaneously  H = change in enthalpy (heat content) T = temperature in Kelvin (  C + 273)  S = change in entropy Free energy  G and chemical reactions

7. Standard free energy (  G  ) and the equilibrium constant When  G is determined under standard conditions of concentration, pressure, and temperature the  G is called the standard free energy change (  G  ) If the pH is set to 7, the standard free energy change is indicated by the symbol  G  ´ A + B ⇄ C + DK eq = [C] [D] / [A] [B]  G  ´ = -RT ln K eq

8. Reactions proceed in the direction of negative  G  ´ Reaction will proceed to the right (downhill process) Reaction will proceed to the left (uphill process)

9. Key issue: how can cells achieve essential reactions with a positive  G  ´? Examples: Nutrient uptake DNA replication Amino acid biosynthesis CO 2 fixation Flagellar motion ATP synthesis

10. By coupling an uphill process to a downhill process A major role of ATP is to drive otherwise endergonic reactions This makes the overall reaction downhill, so it will proceed Free energy input is needed to sustain life and growth Main downhill processes? ATP hydrolysis and proton motive force

11. Energy cycle Note: this is simplification, because it ignores coupling of proton motive force to all three forms of work

12. Adenosine 5´-triphosphate (ATP) ATP serves as the major energy currency of cells “Contains 2 high energy bonds”. Note: there is nothing particularly special about these two bonds except that cells happen to use them. ATP  ADP + P i + Energy P i = orthophosphate Note: ATP is complexed to Mg 2+

13. Oxidation-reduction reactions Oxidation-reduction reactions are key in almost all energy metabolism of life (respiration, photosynthesis, and also fermentation, glycolysis): Coupled to the generation of ATP, proton motive force. Loss of electrons is oxidation (LEO) Gain of electrons is reduction (GER) Aerobic respiration is when O 2 acts as the final electron acceptor (O 2  H 2 O) Acceptor + ne- ⇄ donor, n = number of electrons transferred

14. Quantifying redox reactions 1. Split redox reactions into two half reactions involving two redox pairs. Example: Fe 3+ + Cu +  Fe 2+ + Cu 2+ Fe 3+ + e -  Fe 2+ (electron acceptor) Cu +  Cu 2+ + e - (electron donor) 2. Redox potential  E  (similar to  G  ) =  E  A -  E  D The equilibrium constant of a redox reaction is called the standard reduction potential (E  ).  G  = -nF  E  F=constant of Faraday 2. Define hydrogen half reaction as the absolute reduction reduction potential: 2H+ + 2e- ⇄ H 2 The reference standard for reduction potentials is the hydrogen system with an E  ´ of - 0.42 volts (at pH 7). Note: a positive  E  corresponds to a negative  G  : electrons will flow to the compound with the most positive  E 

15. In our mitochondria: NADH + H + ⇄ NAD + + 2H + + 2e - -0.32 V O 2 + 2H + + 2e - ⇄ H 2 O 0.82 V  E  =  E  A -  E  D so: 0.82 - - 0.32 = 1.14V  G  = -nF  E   G  = -2*23*1.14 = -54.4 kcal/mol ATP hydrolysis: -7.3 kcal/mol Respiration in our mitochondria yields 1.14V of driving force to convert into other forms of energy (pmf)

16. Electrons flow to more positive redox potential Electrons flow from donors with more negative redox potential to acceptors with more positive redox potential.

17. Key electron carriers Electron carriers serve to transport electrons between different chemicals Example - Nicotinamide adenine dinucleotide (NAD) NADH + H + + 1/2 O 2  H 2 O + NAD + NAD + / NADH is more negative than 1/2 O 2 / H 2 O, so electrons will flow from NADH (donor) to O 2 (acceptor)

18. Structure of NAD Water soluble electron carrier

19. Photosynthesis

20. Flavin adenine dinucleotide (FAD) Proteins bearing FAD (or FMN) are referred to as flavoproteins FAD is usually bound to proteins

21. Coenzyme Q (CoQ) or ubiquinone Transports electrons and protons in respiratory electron transport chains. Residues in membrane (hydrophobic molecule) Note: * One-versus two-electron processes * In some cases electron transfer is coupled to protonation/deprotonation

22. Cytochromes Cytochromes are redox proteins that bind a heme. They use the iron atoms in the heme to reversibly transport a single electron Iron atoms in cytochromes are part of a heme group Nonheme iron proteins carry electrons but lack a heme group (e.g. Ferrodoxin)

23. Enzymes Enzymes are protein catalysts * Enzymes catalyze an astonishing array of different reactions * Enzymes speed up reactions without altering their equilibrium position. Note: they can couple down-hill and uphill reactions * Enzymes are permanently chemically altered during catalysis * Enzymes tremendously speed up reactions: typically 10 9 *Enzymes are highly specific Reacting molecules = substrates Substances formed = products

24. Enzymes can have cofactors Some enzymes are composed purely of protein ) Some enzymes contain both a protein and a nonprotein component: a cofactor (like FAD) The protein component = apoenzyme The nonprotein component = cofactor Apoenzyme + cofactor = holoenzyme Cofactor tightly attached to apoenzyme = prosthetic group Loosely bound cofactor = coenzyme

25. Classification of enzymes Enzymes can be placed in six classes and are usually named in terms of substrates and reactions catalyzed.

26. Mechanisms of enzyme activity Central effect: enzymes speed up the rate at which a reaction proceed to equilibrium by lowering the activation energy Activation energy required to from the transition state (AB ‡ )

27. Lock-and-key model Some enzymes are rigid and shaped to precisely fit the substrate(s) Binding to substrate positions it properly for reaction Referred to as the lock-and- key model

28. Induced fit model Some enzymes change shape when they bind their substrate so that the active site surrounds and precisely fits the substrate This is referred to as the induced fit model Glucose binding to hexokinase

29. Describing enzyme activity: K m and V max * Add various concentrations of substrate [S] to a constant amount of enzyme and measure the initial rate V 0 (or v) of the reaction. Question: why the initial rate? * Repeat this for various substrate concentrations and plot V 0 versus [S]. Question: what will the curve look like? And: Where have we seen this curve before?

30. Michaelis-Menten kinetics: K m and V max Hyperbolic dependence of V 0 on [S] Saturation behavior: why?

31. Effect of temperature on enzyme activity Enzymes are most active at optimum temperatures; deviation from the optimum can slow activity and damage the enzyme Question: Where have we seen this before?

32. Effect of pH on enzyme activity Enzymes often have pH optimium. Question: how to explain this? Active site of serine protease Change in protonation state of active site residues. Here: Asp and His. pK a values

33. Enzyme inhibition Many poisons and antimicrobial agents are enzyme inhibitors Can be accomplished by competitive or noncompetitive inhibitors Competitive inhibitors - compete with substrate for the active site Noncompetitive inhibitors - bind at another location

34. Usually resemble the substrate but cannot be converted to products Malonate is competitive inhibitor of succinate dehydrogenase Competitive inhibitors

35. Noncompetitive inhibitors Bind to the enzyme at some location other than the active site Do not compete with substrate for the active site Binding alters enzyme shape and slows or inactivates the enzyme Heavy metals often act as noncompetitive inhibitors (e.g. Mercury)

36. Metabolic regulation Important to conserve energy and resources Cell must be able to respond to changes in the environment Changes in available nutrients will result in changes in metabolic pathways

37. Control of enzyme activity * Allosteric control * Covalent modification

38. Allosteric enzymes Activity of enzymes can be altered by small molecules known as effectors or modulators Effectors bind reversibly and noncovalently to the regulatory site Binding alters the conformation of the enzyme Positive and negative effectors

39. Example: regulation of aspartate carbamyltransferase Regulation of aspartate carbamyltransferase is a well studied example of allosteric regulation CTP inhibits activity and ATP stimulates activity

40. ACTase regulation Binding of effectors cause conformational changes that result in more or less active forms of the enzyme Top view T state Less active R state More active Side view

41. ACTase regulation CTP inhibits activity and ATP stimulates activity Binding of substrate also increases enzyme activity (more than one active site) Velocity vs. substrate curve is sigmoid

42. Covalent modification of enzymes Attachment of group to enzyme can result in stimulation or inhibition of activity Attachment is covalent and reversible Example: phosphorylase b from Neurospora crassa Question: where have we seen this?

43. Feedback inhibition Metabolic pathways can contain a pacemaker enzyme (rate-limiting step) Usually catalyzes the first reaction in the pathway Activity of the enzyme determines the activity of the entire pathway Feedback inhibition occurs when the end product interacts with the pacemaker enzyme to inhibit its activity