Bioenergetics and Metabolism Part II and Chapter 13 Bioenergetics and Metabolism

Bioenergetics and Reactions Key topics: Learning Goals Thermodynamics applies to biochemistry Organic chemistry principles at work Some biomolecules are “high energy” with respect to their hydrolysis and group transfers Energy stored in reduced organic compounds can be used to reduce cofactors such as NAD+ and FAD, which serve as universal electron carriers

Metabolic Pathways Cooperate To: Obtain Chemical Energy by: a. Capturing Solar Energy, or b. Oxidizing Energy Rich Chemicals from the Environment. Convert Nutrient Molecules to metabolic intermediates, then monomers or waste products. Polymerize monomers to polymers (proteins, carbohydrates, nucleic acids, lipids). Synthesize and Degrade (turnover) biomolelcules. The basics of metabolic pathways

Anabolism and Catabolism What is missing: GTP, CTP and UTP. Actually we think in terms of ATP, but all the NTPs and dNTPs are needed. The dNTPs for DNA synthesis, the NTPs for RNA synthesis AND GTP is needed for protein synthesis (translation), CTP is needed for phospholipid synthesis, and UTP is needed for polysaccharide synthesis. Further when ATP or other NTPs are used as a source of energy, they can also be hydrolyzed between the β and γ phosphates (a little more energy than breaking off the terminal γ phosphate) yielding Ppi and a proton (remember Chapter 8). Fortunately the diagram has the major redox players (there is one other we will do in photosynthesis).

Linear and Circular Pathways Pathway architecture: linear converging, linear diverging and circular. The general message is that the catabolism of organic molecules goes towards Acetyl-S-CoA (in Chapter 18 we will see that protein  amino acids  acetyl-S-CoA and a variety of Citric Acid Cycle intermediates, not just acetyl-S-CoA. The diverging linear pathways here are all toward lipids (Chapter 10). In actuality, glycolytic intermediates (between glucose and pyruvate), Pentose Phosphate Pathway intermediates and Citric Acid Cycle intermediates are the ones used to synthesize amino acids, nucleotides, sugars, and vitamins. When these are all put onto paper it looks a bit confusing, next slide

Metabolic Pathways In blackish blue is Glycolysis, Citric Acid Cycle, respiratory electron transport (bottom), but the green to the top intermediate left of glycolysis should be included in the main pathways of Central Metabolism…from which everything comes. That is the human societal analogy: next slide.

Auto-Pathways Are we going the right way yet ???? Pathways are all about flow. Each step is carried out by an enzyme…such that the substrate of an enzyme was the product of the enzyme before it in the pathway. What has evolved to maximize efficiency is that the enzymes are held together in “pathway modules” so that when an enzyme produces a product it doesn’t’ have to float around the cytoplasm in search of the next enzyme….the next enzyme is right there in the module…see the next slide of an excellent cartoon of a section of an E. coli cell.

Pathways Arranged as Multi-Protein Modules Flagella LPS Outer Membrane Peptidoglycan Cytoplasmic Membrane Glycolysis ATPase RNA

5 Main Classes of Metabolic Reactions Oxidation-Reduction Reactions Reactions that Make or Break Carbon-Carbon Bonds Internal Rearrangements, Isomerizations, Eliminations. Group Transfer Reactions. Free Radical Reactions.

Chapter 13 Bionergetics ATP Review

Showed that Respiration Was Oxidation of Carbon and Hydrogen…thus began Thermodynamics Review

Laws of Thermodynamics First Law – for any change, the energy of the universe remains constant; energy may change form or it may be transported, but can not be created or destroyed. Second Law – The Entropy Law can be stated 3 ways: 1. Systems tend from ordered to disordered. 2. Entropy can remain the same for reversible processes but increases from irreversible processes. 3. All processes tend towards equilibrium. Everything  Equilibrium = Death. Third Law – Entropy goes to zero when ordered substances approach absolute zero = 0oK Serious review

Thermodynamics Gibbs Free Energy G and ΔG Enthalpy H and ΔH Entropy S and ΔS ΔG = ΔH - TΔS J.W. Gibbs and “Free Energy”

Biochemistry Uses ΔGo’ Not ΔGo Standard Conditions (all reactants and products at 1M, gases at 1 atm, Temp = 25C) are Not Biological Conditions So, ΔGo’ takes out water (55.5M), and [H+] is set at pH 7 (not 1M which would be pH=0) and for humans ΔGo’ uses 37oC (310 K), but for bacteria ΔGo’ uses 25oC (298 K)….or the temperature of the environment. ΔGo’ = - RT ln Keq You should be able to do EOC Problems 2 and 3 easily EOC Problem 6: the difference between ΔGo’ and ΔG. ΔGo’ has to take out water, 55.5 M would throw off any equilibrium equation when every thing else in in mM or µM or less. When a proton is involved we can not be a pH = 0, so we set it at pH 7. And then some use 25oC and others 37oC….all trying to make it real toward Biology.

Free energy, or the equilibrium constant, measure the direction of processes

Some data. See that hydrolysis of acid anhydrides has more energy than other linkages. At the bottom comparing a sugar getting completely oxidized to CO2 and water (combines Glycolysis, Citric Acid Cycle, respiratory electron transport. Produces lots of energy…but look, palmitate has even more: there are two reasons for that, what are they ?

ΔGo’s Are Additive Hexokinase Rxn: Glucose + ATP  Glucose-6-P + ADP Glucose + Pi  Glucose-P + H2O ΔGo’ = 13.8 kJ/mole ATP + H2O  ADP + Pi ΔGo’ = -30.5 kJ/mole Overall = ΔGo’ = -16.7 kJ/mole Exergonic ! So: K’eq = 7.8 x 102 EOC Problems 9 and 12: the ΔGo’ for 2 coupled reactions.

Biochemical Pathways Have Evolved To: Use reactions that are relevant to metabolic systems: Makes use of available substrates – with reaction rates that are NOT slow (have too high activation energies even with enzymes!) to produce useful products (which are themselves substrates). And, Maximize Rates Evolution’s Toolbox: reactions that work. : circumvent “impossible” reactions. : most reactions in organic chemistry occur in biology, except one, the Diels Alder Rxn…but we will see about that.

You be a radical ! You be inonic ! Review Organic Chem.

Rich in electrons  donate electrons Electron poor  suck up electrons from donors More review.

The Importance of Carbonyls Nucleophile Electrophile Imines are like carbonyls Here the carbonyl is an electrophile

Making and Breaking Single Bonds Review

Isomerations are Internally Complex Thermodynamically neutral or close to it, these reactions can be complex.

Review.

The Classic Redox Reaction This reaction uses NADH or NAD+ depending which way you are going.

ATP Hydrolysis Hydrolysis of acid anhydride bond yields energy. If this was an ester, the energy released would be about -15 to 20 kJ/mole. But being two acid anhydride in a row hydrolyzing it relieves charge repulsion and produces one product that instantly takes one of thee other forms and the other product ionizes at pH 7. So the actual product does not exist and pulls the reaction forward. This in General Biology texts is called a “high energy bond”, is that so? Compare this to the bond energy of most single bonds (Chapter 2). These bonds that are made and broken for energy transfer are really “medium” energy.

Energy Charge [ATP] + ½ [ADP] [ATP] + [ADP] + [AMP] Energy Charge = Most of the nucleotides are in the tri- or di- phosphate form. Energy charge is like finding how well charged up the ATP battery is. It includes half of the ADP because of the enzyme adenylate kinase (next slide). Most living cells can not tolerate an energy charge lower that 0.8; going there or lower is almost certain death. [ATP] + ½ [ADP] [ATP] + [ADP] + [AMP] Energy Charge =

Energy Charge Why the ½ [ADP] ??? It is because of Adenyl Kinase: ADP + ADP  ATP + AMP Which means 2 ADP is equivalent to 1 ATP….therefore ½ of the [ADP] is counted as ATP.

Nucleotide Intracellular Concentrations* Nucleotide Conc, μM Nucleotide Conc, μM ATP 3,000 GTP 923 ADP 250 GDP 128 AMP 105 GMP 20 dATP 175 dGTP 122 dTTP 77 dCTP 65 UTP 894 CTP 515 cAMP 6 cGMP nd ppGpp 31 NAD+ 790 NADP 54 NADH 16 NADPH 146 FAD 51 FMN 88 AcCoA 231 SuccCoA 15 Here you can calculate the energy charge using adenylates and guanylates. in Salmonella enterica subsp Typhimurium from Bochner and Ames, 1982, J. Biol. Chem 257:9759-9769

Magnesium Stabilizes Tri- and Di-phosphates Almost all enzymes using ATP or any NTP requires Mg++ to partly balance the negative charges of the phosphates. EOC Problem 19: How much ATP is used in a human/day. EOC Problem 20: About turn over of the α and β phosphates (can you located them above?).

Pyruvate Kinase This is the last reaction of glycolysis. It is just about the most energetic of these Central Metabolism reactions. It can easily power ATP synthesis from ADP and Pi.

1,3-Bisphosphoglycerate has More Energy Than ATP This is another glycolytic enzyme following the oxidation step in glycolysis, and again it can power the production of ATP and again ionization, resonance stabilization contribute to the forward reaction and having more energy than an ATP acid anhydride bond.

Phosphocreatine Is Store of Energy in Muscle Phosphocreatine is an energy storage molecule in skeletal muscle. It is there to help out in the fight or flight response after the crazy prof yells his head off (Chapter 12). This again for all the right reasons can produce ATP…it helps power only the first few seconds of the fight or flight response…and has been favored in evolution .. So those first few seconds are important.

Note that the hydrolysis of ATP to AMP and PP is the reaction that is part of DNA polymerase…and it has more energy that the normal “medium energy” acid anhydride bond. Acetyl-S CoA is a thio-ester (see slide 37) which has more energy than carbon based esters (all the sugar phosphates).

This is the real, biological ΔG in a cell !! What About Actual ΔG ? ΔG = ΔG’o + RT ln([products]/[substrates]) This is the real, biological ΔG in a cell !! At 25oC RT = 2.48 kJ/mole (2.5 kJ/mole) At 37oC RT = 2.58 kJ/mole (2.6 kJ/mole) We will be doing this a lot later on ! Standard ΔGo’ only show potential. But it is the real ΔG reflecting the actual concentrations of the metabolites in a cell that is important as to whether certain reactions will go in one direction or the other.

Doing Worked Example 13-2 Using E. coli ΔG = ΔGo’ + RT ln [ADP][Pi]/[ATP] ΔG = -30.5 kJ/mole + [ (8.315 J/mole.K)(310K) ln(1.04mM)(7.9mM)/7.9 mM] ΔG = -30.5 kJ/mole + 2.58 kJ/mole (-6.8) ΔG = -30.5 kJ/mole + (-17.6) ΔG = -48.1 kJ/mole Note: Calculate mM such as 1.04mM = 1.04 x 10-3M In the text for the Human Erythrocyte it works out to ΔG = -52 kJ/mole

Acetyl-CoA (Thiol-ester) Has the Energy of ATP! EOC Problem 21: Cleavage of ATP to AMP + PPi…..why is this different (see Table 13-6 above). (What DNA enzyme did the same? It’s in Chapter 8)

Enzyme Reaction Phosphorylation Intermediates Used to form C-N Bonds When we start metabolism, we may look at some interesting mechanisms, but what you need to know is the overall reaction. That is what is important.

Phosphates: Ranking by the Standard Free Energy of Hydrolysis Phosphate can be transferred from compounds with higher ΔG’ to those with lower ΔG’. Reactions such as PEP + ADP => Pyruvate + ATP are favorable, and can be used to synthesize ATP. FIGURE 13–19 Ranking of biological phosphate compounds by standard free energies of hydrolysis. This shows the flow of phosphoryl groups, represented by P, from high-energy phosphoryl group donors via ATP to acceptor molecules (such as glucose and glycerol) to form their low-energy phosphate derivatives. (The location of each compound’s donor phosphoryl group along the scale approximately indicates the ΔG’° of hydrolysis.) This flow of phosphoryl groups, catalyzed by kinases, proceeds with an overall loss of free energy under intracellular conditions. Hydrolysis of low-energy phosphate compounds releases Pi, which has an even lower phosphoryl group transfer potential (as defined in the text).

Nucleoside Diphosphate Kinase makes NTP’s from ATP and NDP’s This simply means that all the nucleotide phosphates can interchange their phosphates.

Carbon Redox – Watch the Red Dots (Electrons) Reduced to oxidized…review

Emf or Eh or Eo Redox review.

All standard half cells are written as reductions All standard half cells are written as reductions. Review your redox chemistry. The more negative the value, the more energy to donate electron(s) to a more oxidized acceptor. EOC Problem 24: Respiratory chain thermodynamics (we will do this in Chapter 19)…learn it well now!

Calculations Differences between half cells…Example of electron transfer from NADH to cytochrome-b: NADH Eo’ = -.32 v Cyt-b Eo’ = 0.077 v ΔEo’ = Eo’oxidized – Eo’ reduced = 0.077v – (-0.32v) ΔEo’ = 0.397v NADH is commonly produced from oxidative reactions putting the two electrons onto the nicotinamide portion of NAD+ to reduce it to NADH. Here NADH is donating the electrons to reduce cytochrome-b.

Further Calculations What is the ΔG’o for oxidation of NADH by cytochrome-b ΔG’o = - nℱ ΔEo’ Faraday Constant = 96,480 J/v.mole ℱ = 96.5 kJ/v.mole ΔG’o = - (2) 96.5 kJ/v.mole (0.397v) = - 77 kJ/mole What about the real ΔE ?...and then ΔG ! Getting from ΔEo’ to ΔG’o ΔE = ΔE’o + (RT/nℱ) ln ([products]/[substrates]) EOC Problem 25 and 26 are all about this.

NAD+ + 2e- + 2H+  NADH + H+

This is a fairly trivial, but real point.

Lactic Acid Dehydrogenase = LDH Rossmann fold, a structural motif in Dehydrogenases

Vitamin Niacin is Made from W and Needs to be Amidated for NAD+

FMN and FAD Reduced flavins are less stable than reduced NADH and NADPH. But they are still used.

Yes all these electron carriers are real.

Enzyme Reactions have a Yield of ~1.0 The Perfect Catalysts Assume Metabolism worked on each step in the metabolic pathway having a yield of 0.9 (tremendously high for organic chemistry reactions!!). Then look at a 10 reaction pathway such as Glycolysis: if you start with 100 mg of glucose the pathway would only produce less than 39 mg of pyruvate….AND, the cell would fill up with 61 mg of side reaction products!  The message is that metabolism and life would be tremendously inefficient = motionless, wasteful BLOBS filled with junk. Most enzymes have a yield of 0.9990 to 0.99990 (that is they make a mistake reaction 1 in a 1,000 to 10,000 reactions). It is likely a reason why metabolism took billions of years to get to larger life forms.

What is the BEST Yielding Enzyme? Answer: DNA polymerase makes an error 1 in 107 to 109 reactions. Why? It is the one of the few enzymes to have a “proof- reading” function to correct the 1 in 103 to 104 mistakes. Check it out in Molecular Biology! DNA polymerase is fast and accurate.

Things to Know and Do Before Class* The basic laws of thermodynamics. Be able to calculate ΔG, ΔGo’ from concentrations or Keq. Be able to calculate over all ΔGo’ from summed reactions. Principles that make some bonds “high energy”. EOC Problems (2, 3), 6, 7, 9, 12, 14, 20, 21, 24-26. *Two Class periods for this chapter.