1. Hemoglobin and Myoglobin
Myoglobin (Mb) : stores O2
Hemoglobin (Hb) : transports O2
Each of the four chains in Hb looks like Mb, and each carries a heme.
Hemoglobin contains two identical a chains and two identical b chains. Letters A–H are a-helical regions.
The protein 1) protects heme from oxidation and 2) provides a pocket where only O2-like molecules can fit

2. The geometry of iron coordination in oxymyoglobin:
The octahedral coordination of the iron ion. The iron and the four nitrogens from protoporphyrin IX lie nearly in a plane.
A histidine (F8, or His 93) occupies one of the axial positions, and O2 the other.
Schematic drawing of the heme pocket, showing the proximal (F8; His93) and distal (E7; His64) histidine side chains.

3. UV-Vis spectrum of Heme Proteins
Changes in the visible spectrum of hemoglobin.
Spectra for hemoglobin in the deoxygenated state (blue trace) and the O2-bound state (red trace) are shown.
Hemoglobin in the deoxygenated state is a venous purple, whereas completely oxy-Hb is bright red.
As more O2 binds to Hb, the visible spectrum shifts from the blue to the red trace

4. Binding of O2 by Mb & Hb
Fe moves 0.3 Ǻ back toward plane of heme

5. Binding Curves
K = equilibrium association constant

6. Binding Curves are ubiquitous in science & medicine
Some Examples…
Dose-response curves
Ligand (drug) / receptor binding
curves
Enzyme/substrate activity curves

7. Oxygen Transport: why Hb works
Efficiency in O2 transport is achieved by cooperative binding in multisite proteins, described by a sigmoidal (S-shaped) binding curve. Sigmoidal curves are also seen for DNA melting, enzyme activity)

8. The conformational change caused by O2 binding
Many salt bridges and H-bonds are broken and relocated upon conversion for deoxy to oxy
What happens in between? (how does the allosteric change happen?)
Above: salt bridges in deoxy Hb. Note the importance of acid-base chemistry

9. Conformational changes in Hb during the deoxy-oxy transition
Oxygenation leads to a 15° rotation of one αβ dimer with respect to the other. Note also how the inner pocket changes shape.

10. Bohr Effect How does this work?
For example, in muscle tissue, the pH is lower (more acidic) than in the lungs. The higher [H+] helps to drive O2 unloading in the capillaries.

11. Binding of BPG lowers affinity of Hb for O2

12. Sickle-cell anemia Normal Hb  normal erythrocyle
Hb S  sickled erythrocyte
hydrophobic pimple from Glu  Val mutation at β6
Hb S forms a long fibrous polymer of linked Hb S
One aa change  molecular disease…REMARKABLE!
Sickle-cell anemia confined to people originating in tropical area because people with α2ββ(s) are more resistant to malaria

13. Enzymes: Transition State Theory
Transition State (T.S.) : high energy species that is formed transiently en route from reactants to products
Free energy of reaction (ΔGo): difference in free energies of reactants and products.
Free energy of activation(ΔG‡): energy difference between reactants and the top of the hill, i.e. the highest energy T.S. ΔG‡ is also called the activation energy (Ea).

14. How do enzymes work? Ea determines the kinetics of a reaction.
If Ea is large  reaction is slow
If Ea is small  reaction is fast
Enzymes speed up reactions by lowering the activation energy (the height of the hill)
Note: the thermodynamics (ΔG0) of the reaction
are unchanged – the enzyme only speeds up the reaction

15. Enzyme – Substrate Complexes
Substrate: reactant in the reaction catalyzed by the enzyme
Enzymes bind substrates in a specific location, called the active site, to form enzyme substrate complexes (ES complexes)

16. Stereospecificity conferred by an enzyme:
Active sites have 3-D structure
Chiral substrates bind specifically to the active site
(ex. Epinephrine)
The asymmetric surface of an enzyme can even confer stereospecific binding with a symmetric substrate.

17. 2 Models for Enzyme-Substrate Interactions
Lock and Key: active site of enzyme is complementary in shape to the substrate
Enzyme Substrate = ES
A static model

18. 2 Models for Enzyme-Substrate Interactions
Induced Fit: Active site not rigid
Shape modified through binding of substrate
Shape of active site complementary to that of the substrate only after substrate is bound
Enzyme Substrate = ES
A model of dynamic recognition

19. Introduction to Enzymes
Enzymes are very selective about which substances they interact with and which reaction they catalyze.
This specificity, along with the regulation of enzymatic activity, is what allows the many diverse metabolic pathways to be harmoniously interconnected for life.

20. Michealis Menten Kinetics
Enzyme activity (or velocity V) can be modulated by:
substrate concentrations
phosphorylation
allosteric effectors (like hemoglobin)

21. Example: Hexokinase
The binding of glucose to hexokinase induces a significant conformational change in the enzyme.
The enzyme is a single polypeptide chain, with two major domains.
Notice how the obvious cleft between the domains (panel a) closes around the glucose molecule.
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22. Serine Proteases

23. Chymotrypsin structure
The structure of chymotrypsin and the serine protease catalytic triad:
The backbone of bovine chymotrypsin detemined by X-ray crystallopgraphy.
Here, H57 is protonated, S195 and D102 are deprotonated.
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24. Chymotrypsin mechanism (1)
Catalysis of peptide bond hydrolysis by chymotrypsin:
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25. Chymotrypsin mechanism (2)
Catalysis of peptide bond hydrolysis by chymotrypsin:
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27. Linear Plots: Lineweaver-Burk

28. Linear Plots: Eadie-Hofstee
Graphing v versus V/[S], we obtain Vmax at (V/[S]) = 0 and KM from the slope of the line.

29. Enzyme Inhibition: Competitive

30. Enzyme Inhibition: Noncompetitive

31. Fast Reactions
Stopped-flow
Pushing triggers data collection begins. Mixing takes ~ 1 ms, so limited to enzymes with kcat < 103 s-1 31

32. Temperature Jump
For really fast kinetic processes (sub-millisecond processes)
Say reaction mixture is at equilibrium at T1 …
Quickly increase T1 to T2 by passing current between electrodes in the mixture, then watch relaxation to new equilibrium
∆ [A](t) = (∆ A)tot e-t/
= relaxation time  gives rate constants
1/  = k1 + k-1
For 32

34. Contributions of various His residues to the Bohr effect
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