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pH and fumarase

pH and fumarase. Forward reaction: B 2 has to accept a proton from water What if pH is too low? What if pH is too high?. This week’s lab notes. You want to know the total activity of each fraction slope ( D abs/min) → rate ( m mol/min)

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pH and fumarase

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  1. pH and fumarase Forward reaction: B2 has to accept a proton from water What if pH is too low? What if pH is too high?

  2. This week’s lab notes • You want to know the total activity of each fraction slope (Dabs/min) → rate (mmol/min) Think of this rate as # units of fumarase activity in the volume you assayed (eg. you may have added 10 mL to 990 mL assay buffer). But, you have to correct for the total volume of the sample. (eg. you may have applied 10.4 mL of crude to the column)

  3. How much of that sample you tested for activity (~10mL) From Dabs/time Sample’s total activity vs. crude’s

  4. Plan: • Exam over Ch. 4, 5.1 plus Expt 3 weeks 1 and 2 (fumarase purification and ion exchange) • Today: finish up 5.1 (Hb), start Ch. 6

  5. Hemoglobin • Cooperative binding • Binding of O2 at one subunit affects the oxygen affinity of other subunits • Allostery: • Regulation by reversible binding at a site other than the active site • “Allosteric activation” • O2: homotropic allosteric activator

  6. Another allosteric modulatorbisphosphoglycerate (BPG) • Heterotropic allosteric inhibitor • Binding of Hb•BPG has a lower affinity for O2 than does Hb • Enhances release of O2 in the tissues

  7. One BPG molecule per tetramer Pushes T ↔ R equilibrium to the left

  8. T state High affinity for BPG Stabilized by BPG Low affinity for O2 R state High affinity for O2 Stabilized by O2 Low affinity for BPG

  9. Enzymes • Biological catalysts • High specificity and efficiency relative to inorganic catalysts, for example • Participate in reactions, but no net change • Lower the activation energy • Do not change equilibrium (get there faster)

  10. Enzymes • Almost exclusivelyproteins (some RNA, others?) • Protein may require cofactor(s) (non-amino acid functional groups) • Apoenzyme: protein alone • Holoenzyme: protein + functional group • Metals, nucleotide-containing cofactors, etc.

  11. Enzymes • Usually noted by “-ase” at the end • DNA polymerase, protein kinase, etc. • Many enzymes have a common ‘trivial’ name • Fumarase, hexokinase, lysozyme, etc. • All enzymes have a systematic name • Substrate(s) and reaction catalyzed • Fumarase = “fumarate hydratase” • Hexokinase = “ATP:glucose phosphotransferase”

  12. Enzymes • Some common classes of enzymes • Kinases transfer phosphate (usually from ATP) to another substrate • Phosphatases remove (hydrolyze) a phosphate • Polymerases string together nucleotides • Proteases cleave peptide bonds • Oxidoreductases transfer electrons between substrates

  13. aspirin Drugs often modulate the action of enzymes Arachidonic acid Prostaglandin H2 CYCLOOXYGENASE www.3dchem.com

  14. Enzymes speed up biological reactions H2CO3 → CO2 + H2O 10,000,000x faster + carbonic anhydrase

  15. Biological reaction: sugar + oxygen ↔ CO2 + water High energy “Transition state” Intermediate between R & P Activation energy EA Kinetic barrier to reaction ENERGY (G°) DG < 0 Reactants (R) Products (P) Reaction should be spontaneous Equil should favor products REACTION PROGRESS

  16. The energy barrier is critical for life • Potentially deleterious reactions are blocked by EA • Complex molecule degrading to simpler constituents nucleotide DNA http://asm.wku.edu http://encyclopedia.quickseek.com/

  17. How do enzymes speed up reactions? • Lower the activation energy • Decrease the energy barrier 2H2O2→ 2H2O + O2 Hydrogen peroxide Isolated: EA ~ 86 kJ/mol In the presence of catalase: EA ~ 1kJ/mol

  18. Binding of substrate to enzyme creates a new reaction pathway Without enzyme With enzyme EA = DG‡ An enzyme changes EANOTDG Affects RATE, not EQUILIBRIUM http://w3.dwm.ks.edu.tw/

  19. How is EA lowered? EA = DG‡ = DH - TDS entropy enthalpy • Enzyme’s ‘goal’ is to reduce DG‡ • Two ways enzymes can affect DG • Improve DH • Improve DS DG‡ = Gtrans.state – Greactants Enzymes alter the free energy of the transition state

  20. BH+ BH+ A A - Example: More favorable DH Charge unfavorable Unstable transition st. OH- + OH- B H2O + A Ionic interaction stabilizes the positive charge BH + AOH

  21. Example: More favorable DS One molecule Lower disorder (low S) Unfavorable entropically Two molecules More ‘freedom’ Higher disorder (high S)

  22. Example: More favorable DS ENZYME ENZYME Enzyme/Transition state complex Still a single molecule Not much difference entropically Enzyme/Reactant COMPLEX Essentially a single molecule

  23. Remember • Enzymes lower the energy barrier • Decrease EA (DG‡) • Provide an environment where: • Transition state is stabilized (lower enthalpy) • Change of disorder (entropy) is minimized

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