1. 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?

2. This week’s lab notes You want to know the total activity of each fraction slope (  abs/min) → rate (  mol/min) Think of this rate as # units of fumarase activity in the volume you assayed (eg. you may have added 10  L to 990  L 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. From  abs/time Sample Total Volume (mL) Rate (  mol/min) Volume Assayed (mL) Total Activity (  mol/min) Yield (%) Crude10.40.250.010260100 FT14.10.050.01070.527.1 Pooled Elutions 3.20.310.01099.238.2 How much of that sample you tested for activity (~10  L) 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 O 2 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” –O 2 : homotropic allosteric activator

6. Another allosteric modulator bisphosphoglycerate (BPG) Heterotropic allosteric inhibitor Binding of HbBPG has a lower affinity for O 2 than does Hb Enhances release of O 2 in the tissues

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

8. T state R state High affinity for BPG Stabilized by BPG Low affinity for O 2 High affinity for O 2 Stabilized by O 2 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 exclusively proteins (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. Drugs often modulate the action of enzymes CYCLOOXYGENASE aspirin www.3dchem.com Arachidonic acid Prostaglandin H 2

14. Enzymes speed up biological reactions H 2 CO 3 → CO 2 + H 2 O 10,000,000x faster + carbonic anhydrase

15. ENERGY (G°) REACTION PROGRESS  G < 0 Reaction should be spontaneous Equil should favor products Biological reaction: sugar + oxygen ↔ CO 2 + water Reactants (R) Activation energy E A Kinetic barrier to reaction High energy “Transition state” Intermediate between R & P Products (P)

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

17. How do enzymes speed up reactions? Lower the activation energy Decrease the energy barrier 2H 2 O 2 → 2H 2 O + O 2 Isolated: E A ~ 86 kJ/mol In the presence of catalase:E A ~ 1kJ/mol Hydrogen peroxide

18. Binding of substrate to enzyme creates a new reaction pathway http://w3.dwm.ks.edu.tw/ An enzyme changes E A NOT  G Affects RATE, not EQUILIBRIUM Without enzyme With enzyme E A =  G ‡

19. How is E A lowered? Enzyme’s ‘goal’ is to reduce  G ‡ Two ways enzymes can affect  G –Improve  H –Improve  S E A =  G ‡ =  H - T  S  G ‡ = G trans.state – G reactants Enzymes alter the free energy of the transition state enthalpy entropy

20. - Example: More favorable  H A B AOH BH A BH + + H2OH2O +OH - + Charge unfavorable Unstable transition st. A BH + Ionic interaction stabilizes the positive charge OH -

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

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

23. Remember 1.Enzymes lower the energy barrier 2.Decrease E A (  G ‡ ) 3.Provide an environment where: Transition state is stabilized (lower enthalpy) Change of disorder (entropy) is minimized