HB/MB BIOCHEM NOTES (exam 2 pt 2)

Hemoglobin vs Myoglobin

• Myoglobin (Mb) - monomer

  • O₂ transport/storage in muscle

  • 8 alpha helices A-H

  • hyperbolic curve

  • p₅₀ = 2.8 torr

• Hemoglobin (Hb) - tetramer

  • transports O₂ from lungs to tissues; found in blood

  • 4 Mb-like subunits (2 alpha and 2 beta)

  • dimer of dimers (ab)₂ dimer; break dimer with urea

  • sigmoidal curve (factors in T and R state; cooperativity)

  • p₅₀ = 26 torr

• Very similar 3D structures

• Both have 8 alpha helices

• Hb sigmoidal curve = proof of cooperativity

Who discovered Hb structure

PERUTZ

Structure: Heme = protoporphyrin IX + Fe2+​

• protoporphyrin IX = ring shaped “holder” of Fe2+

• Fe2+ = center of ring; What binds to O₂

Noticed crystal structure of oxy/deoxy Hb differed due to quat. level (15 degree twist between dimers)

• Deoxy - T state (low affinity O₂)

• Oxy - R state (high affinity O₂)

Explain octahedral coordination complex (remember E above F)

• Octahedral coordination complex

  • proximal His F8 at bottom of iron

  • distal His E7 forces O₂ to bind at bent angle (reversible)

What to know about CO poisoning

CO (carbon monoxide) has high affinity for Fe2+ (esp linearly)

Distal His E7 makes CO bent → reduce affinity from 20k to 200 better than O₂

what is methemoglobin (metHb) and metmyoglobin (metMb)

when Fe2+ gets oxidized in Mb or Hb → Fe3+ (O₂ cant bind); brownish color

need diaphorase to reduce Fe3+ → Fe2+

Mb O₂ binding equation/fractional saturation (know 2.8 torr)

pO₂/(p₅₀+pO₂) = pO₂/(2.8+pO₂)

intense exercise → low pO₂ → Mb release oxygen

What does higher p50 mean?

Lower affinity for O₂ (shifts curve to the right)

Perutz mechanism (T→R transition comes from Fe-O₂ bond energy formation)

T state (His E7 and Val E11 difficult O₂ binding; Fe out of heme plane) → O₂ binds → His F8 pulls F helix towards heme plane (15 degree rotation to R state) → His E7 and Val11 move out of the way (easy O₂ binding)

a1-b2 and a2-b1 interact

why is Hb a good transport for O₂

In lungs: high pO₂ → R-state (load up on O₂)

In capillaries: low pO₂ (30 torr) → T-state (releases O₂ bc low affinity)

Activators vs Inhibitors

Activator = O₂ (homotropic)

Inhibitor = BPG, H, CO₂, Cl-

Bohr effect

increase [CO₂] or temp → release O₂ (decreased Hb affinity/ lower on curve)

CO₂ binds to N terminal amino group → carbamate in T state

result of N Terminal amino group and His146

Causes imidazole ring to be protonated (in T State)

What state are Bohr protons in?

Bohr protons = T state

change in state → change in pKa → H+ released → O₂ better held (R state)

carbonic anhydrase

• Catalyzes CO₂ + H₂O → H₂CO₃ rx

• Zn polarizes water to make it better nucleophile → then O attacks Co₂ →

• lyase

• part of Bohr Effect

2,3-biphosphoglycerate (BPG)

• shifts eq towards T state (only binds to T)

• makes Hb P₅₀ so high (26 vs 12)

• useful at high elevations (more bpg → better O₂ delivery)

MWC concerted vs KNF induced fit

• MWC - subunits are either all T or all R; as more ligand binds → R state favored

  • only show pos cooperativity

• KNF - more accurate; subunits can be T or R (variation); conformational change induces neighbors (all subunits gradually become R);

  • better bc accounts for pos and negative cooperativity

What is the sickle cell (HbS) mutation

Glu replaced by Val on residue of beta chains

In T state, HbS polymerize → sickle

why HbF higher O₂ affinity than HbA

sequence is different (ser replaces His143→ lower affinity) also has lower affinity for BPG

Chymotrypsin specific for

deep hydrophobic pocket (aromatic substrate); F, W, Y

Irreversibly inhibited by TPCK (think PC - chymotrypsin)

Trypsin specific for

deep negative pocket (positive substrate); Arginine and Lysine (R and K)

Irrevisibly inhibited by TLCK

Elastase specific for? What are all ser protease inhibited by

V, A, G, S

All Ser proteases inhibited by diisopropyl fluorophosphate (DIFP)

RNAse mech info

• His 12 (top, base)

• His 119 (bottom, GA)

• 2’,3’ cyclic nucleotide intermediate

Serine Protease mech info

• Asp 102 - electrostatic stabilization

• His 57 - GABC

• Ser 195 - covalent catalysis

• tetrahedral intermediate with oxyanion hole

• acyl enzyme intermediate

Lysosyme mech info

• D-NAM and E-NAG

• Glu35 (top) - GABC

• Asp52 - covalent catalysis (→ covalent intermediate)

• oxonium ion transition state

Michaelis Menten Equation

inital v = Vmax[S]/(Km+[S])

what is Vmax? Vmax equation?

Vmax = when enzyme substrate is saturated

Vmax = kcat[E_t] (E_t = enzyme total)

competitive inhibitor (active site)

• looks like substrate

• DOES NOT CHANGE Vmax (can be outcompeted by substrate)

• Km increases (affinity decreases)

• alpha = km’/km

Mixed/Noncompetitive Inhibitor (bind to E, not active site)

• Decreases Vmax (higher y-int)

• Km stays the same

• a = Vmax/Vmax’

  • note: for alpha, just divide bigger/smaller to get number > 1

Uncompetitive Inhibitor (only ES complex)

• V_max decrease (Vmax/a)

• Km decreases (Km/a) so affinity for S increases

• SLOPE IS THE SAME FOR BOTH LINES

How to calculate KI

KI = [I]/(a-1)

Need to use LB plot

Group Specific Reagent

• Irreversible Inhibitor

• modifies essential R group

  • ex. iodoacetate affects thiol side chain → Heavy metals also bind to thiol → inactivate enzyme (toxic)

reactive substrate analog

• Irreversible Inhibitor

• resemble substrate and bind at active site

• irreversibly modify/bind crucial functional group

  • ex. TPCK (His 57 of chymotrypsin) and TLCK (His 57 trypsin) 

Suicide Inhibitor

• Irreversible Inhibitor

• resemble substrate but not active at the start; chemically inert in absence of enzyme

  • good drug candidates bc low side effects

  • I -> I* which reacts with E

Bi Bi Reactions

• ordered sequential 

• random sequential 

• Ping pong (double displacement) - A and B never come into contact

  • A transfers functional group to E (E->F) and leaves as P; F transfers fg to B (B->Q); after F transfers fg, changes back to E

• V max increases so increase in rate of rxn

Regulatory strategies

• Control [enzyme] (slow) - synthesis and degradation

• Control enzyme activity -  protein-protein interaction (ex. trypsin inhibitor), allosteric regulation, post translational modification (ex. zymogen activation), reversible covalent modification

What AA are most affected by kinases

Ser, Thr, Tyr (OH groups), and His