Biochemists define standard conditions

1. Biochemists define standard conditions • Physical constants are calculated based on certain standard state parameters: • [water] = 55.5 M • [H+] = 10-7 M (pH of 7) • [Mg2+] = 1 mM • Temperature = 298 K or 25oC • Pressure = 1 atm • Reactants and products have initial concentrations of 1 M

2. Standard Free energy change is related to the equilibrium constant DG’o = -RT lnK’eq Standard free energy is another way of expressing a reaction’s equilibrium constant

3. Actual Free energy depends on reactant and product concentration DG = DGo + RT ln [Products]/[Reactants] DGo is a constant calculated under standard conditions However, biochemical reactions do not necessarily occur under standard conditions the terms in red denote the prevailing conditions of the reaction DG determines if a reaction is favorable

4. Free energy change is independent of pathway • Diamonds are unstable but the transition is slow

5. Add DGo for sequential reactions • Thermodynamically unfavorable (endergonic) reactions are driven by coupling them to favorable reactions • Glucose + Pi  Glucose-6-P + H2O DGo = 13.8 kJ/mol ATP + H2O  ADP + Pi DGo = -30.5 kJ/mol Glucose + ATP  Glucose-6-P + ADP DGo = -16.7 kJ/mol

6. ATP is currency for cellular energy • Energy stored in the bonds of ATP is often utilized in metabolism to drive unfavorable reactions • Although hydrolysis of Mg-ATP is highly exergonic, the activation energy is quite high resulting in a stable pool of Mg-ATP

7. Free energy of ATP hydrolysis • Although DGo = -30.5 kJ/mol for ATP hydrolysis, DG is actually more like –50 to –65 kJ/mol • This is due to differences in ATP, ADP, and Pi concentrations • Maintaining a higher concentration of ATP increases the available free energy • Protein binding affects the concentration of free ATP, ADP, and Pi

8. Additional compounds can act as energy donors • Phosphorylated compounds such as PEP, phosphocreatine, etc. • Thioesters – CoA

9. How does ATP provide energy? • It’s not just hydrolysis, although that does drive conformational changes in proteins such as myosin and kinesin • The “high energy” of ATP stems from the products of reactions involving ATP hydrolysis having a smaller free-energy content than reactants • ATP is a prime example of an energy “shuttle”

10. Phosphate transfer activates metabolites • Transfer of a phosphoryl group puts free energy into the resulting compound • For instance, the phosphorylation of glucose activates this compound in glycolysis

11. Various enzymes facilitate phosphoryl transfer • All known as Kinases • Ie. Adenylate kinase (DGo ~0) • 2 ADP  AMP + ATP • Some also described as phosphatases • Ie. Inorganic pyrophosphatase or alkaline phosphatase

12. How does it “start”, how do cells make ATP? • That discussion begins with an explanation of Biological oxidation-reduction reactions

13. Chemiosmotic mechanism • Cells begin with a reduced compound such as glucose, successively oxidize that compound to release electrons to a membrane bound electron transport chain • Electron flow is coupled to generation of a trans-membrane proton gradient • A specific enzyme ATP synthase uses the resulting protonmotive force to make ATP

14. Electron transfer mechanisms • Directly as electrons, for example cytochrome-mediated electron tranfer (Fe) • As hydride ion (H-) • Combination with oxygen • And dehydrogenation

15. 2 H+ + 2 e- 2 H+ + 2 e- Electrons are often liberated through dehydrogenation HCOOH CO2

16. Potentials between electron donor and acceptor • E’o is the standard reduction potential (pH 7) • Used to predict which direction electrons will flow (more positive means stronger tendency to acquire electrons)

17. The Nernst equation • E = E’o + RT/nF ln ([electron acceptor]/[electron donor]) • This equation allows determination of reduction potentials under nonstandard conditions • E is the reduction potential • n is the number of electrons transferred • F is the Faraday constant

18. Reduction potentials are related to free energy changes DG = -nFDE Or DG’o = -nFDE’o The transfer of electrons constitutes a electronmotive force that can provide energy for doing work

19. When you eat a cookie… • Glucose is enzymatically oxidized, electrons flow through a series of electron carriers to other chemical species, such as oxygen • This electron flow is exergonic, because oxygen has a higher affinity for electrons than do other electron carriers.

20. Cells utilize diverse electron carriers aa shuttles • NAD, NADP, FMN and FAD are water-soluble electron carriers that serve as “coenzymes” • NAD and NADP are highly mobile, while FMN and FAD typically remain enzyme bound • Quinones, such as ubiquinone and plastiquinone, are lipid-soluble and move within the membrane environment

21. More cellular electron carriers • Cells synthesize specific proteins to facilitate electron trasfer between other cellular proteins • Cytochromes – heme and FeS containing proteins • Ferredoxins – FeS containing protein

22. NAD and NADP

23. Some points of interest concerning NAD and NADP • Absorbance at 340 nm • Accepts hydride ion (2 electrons/1 proton) • In cells, typically 10X NAD vs. NADP, and found mostly as NAD and NADPH • NAD typically functions in oxidations (electron acceptor); NADPH functions as coenzyme in reductions • Pyridine nucleotide transhydrogenase- a specific enzyme that monitors pools

24. Flavoproteins bind FAD

25. Some FAD points of interest • FMN – flavin mononucleotide • FAD – Flavin adenine dinucleotide • Sequentially accepts protons and electrons, can maintain one proton and one electron in semiquinone state, or two electrons and two protons in fully reduced state • Reduced state have absorption maximum around 450 nm • Does not diffuse in cells, electron transfer more complicated