BIBC 100 Final Review Session

1. BIBC 100Final Review Session (Final Saturday 8-10:50am)

2. Enzymes • Biological catalysts that lower the activation energy of a reaction • Favors/stabilizes intermediate formation over substrate • Michaelis-Menten constant (Km) indicates [S] at which vo = ½ Vmax (vo is initial rate of product formation for constant [E] at variable [S]) • vo = (Vmax x [S]) / (Km + [S]) • Lineweaver-Burk plot is a reciprocal of MM equation, using straight lines instead of hyperbola 1/vo = (Km/Vmax) x (1/[S]) + 1/Vmax

3. Serine Protease • Catalytic triad Ser, His, Asp cuts polypeptide at a specific peptide/scissile/amide bond (ex. Chymotrypsin at bulky aromatic groups) • Main chain substrate binding—selects protein as a substrate (binds backbone) • Specificity pocket—recognize specific side chains and sequences • Oxyanion hole—stabilize transition state over normal substrate state • Catalytic triad—form tetrahedral intermediate, hydrolyze the peptide bond

4. Serine Protease • Reaction • Peptide bond enters active site • Nucleophilic attack by Serine hydroxyl to C=O of peptide bond • Tetrahedral intermediate formation, stabilized by oxyanion hole • Peptide bond cleavage, peptide-N-terminal half leaves, acyl-enzyme intermediate left in active site • Water enters, His removes H+ from water, nucleophilic attack by water OH- forms second tetrahedral intermediate • Formation of peptide-C-terminal and reformation of Serine hydroxyl using H+ from His, peptide-C-terminal half leaves

6. Membrane Transporters • Channels facilitate diffusion (ex. KcsA K+ channel) • Down/with concentration gradient • Does not require energy to transport • Similar to enzymes in that they lower energy cost to cross the membrane • Pumps require energy (ex. SERCA pump) • Up/against concentration gradient • Requires energy, ex. ATP • Transmembrane domains, identifiable with hydropathy plot (hydropathy index >1 at consecutive transmembrane residues in α-helices) • Nicotinic acetylcholine receptor (nAChR) and bacterial KcsA K+ channel

7. nAChR • Ligand-gated cation channel in skeletal muscle cells at the neuromuscular junction (NMJ), 5x 4-α-helical bundle α2βγδ with m2 helix facing the pore • Acetylcholine binding to 2x α subunits causes conformational change and V/L hydrophobic residues in m2 helix swing outward, opening the channel • Glu- ring on outer membrane side of channel causes electrostatic gating, preventing anions from crossing • Hydrated Na+ions pass through channel because of gradient

8. KcsA K+ Channel • Bacterial Potassium membrane channel, 4x 3-α-helical bundle • Only allows K+ to pass through (from cytosol to extracellular side, with/down the [K+] gradient) • Open state has aqueous cavity opened to cytosolic side • K+ hydration shell is stripped so it can enter narrow selectivity filter from the aqueous cavity. Backbone C=O of peptides in selectivity filter form a carbonyl shell around the K+ ion. Since the energy required to break hydration shell is supplied by energy created from forming C=O---K+ hydrogen bonds, the movement of K+ is driven by concentration gradient • Na+ cannot pass through because it is of the wrong distance from C=O in the selectivity filter, forming weaker bonds that don’t release enough energy to strip the Na+ hydration shell

9. Calmodulin • Calcium-modulated protein (CaM) with 4x EF-hand motifs (2x α-helices) that bind up to 4 Ca2+ ions • Linker α-helix is actually loosened and not a helix when CaM forms a hydrophobic clamp around a target peptide • Activated by Ca2+ ions binding, then activates other proteins • 4Ca2+/CaM-MLCK – Activates MLCK which (+P) MLC which allows crossbridge formation -> contraction in smooth muscles • 2Ca2+/CaM-K+ - CaM-activated K+ channels allows repolarization of depolarized membrane of neuronal/muscular cells • 2Ca2+/CaM-EF – CaM-activated anthrax oedema factor from anthrax bacteria activates cAMP second messenger cascade without cessation, causing massive exocytosis and salt loss, followed by water loss, diarrhea, dehydration, and death • Ca2+/CaM-activated Ca2+ pumps on plasma membrane and sarcoplasmic reticulum keep intracellular/cytosolic [Ca2+] low to allow muscle contraction/relaxation cycles and calcium waves

10. Myoglobin • Monomeric globin protein in heart, skeletal muscles that act as a reserve of oxygen • Like Hemoglobin, has heme group with ferrous Fe2+ ion covalently and h-bond linked to porphyrin ring. Ring is stabilized by hydrophobic interaction • Very high affinity for oxygen at physiological range, enzymatic binding • pO2 arterial/lungs is 100 Torr • pO2 venous/tissues is 40 Torr • pCO2 arterial/lungs is 40 Torr • pCO2 venous/tissues is 46 Torr

11. Hemoglobin • Tetrameric (αβ)1(αβ)2 globin protein • Allow rapid diffusion of O2 into blood, transport O2 through circulatory system • High affinity for O2 at lungs/arterial side (~90% sat) but markedly lower affinity for O2 at tissues/venous side (~75% sat) • Gives up O2 to myoglobin at tissues • Gives up O2 to fetal Hb, which has higher aff. • Allosteric regulation • Bohr effect—increasing acidity, lower pH (from CO2->HCO3-) decreases Hb aff. for O2 • 2,3DPG at high altitude binds to center of tetramer, lowers aff. • Increasing temperature lowers affinity • CO binding radically increase affinity, leading to suffocation

12. Cooperativity • Hill plot is a logarithmic scale plot of fraction saturation versus pO2. Hill coefficient is slope of binding curve at 50% sat. • Hill coefficient of 1 indicates no cooperativity (ex. Myoglobin) • Hill coefficient of >1 indicates positive cooperativity (ex. Hb ~3) • Hill coefficient of <1 indicates negative cooperativity • Binding of O2 pulls Heme group and F helix toward E helix of a subunit, changing subunit from T (tense/deoxy) to R (relaxed/oxy) state. Axis of (αβ)1 dimer tilts in relation to (αβ)2 dimer, weakening α1-β2and α2-β1 h-bonds between dimers. • Symmetric model of cooperativity has 5 states of O2 binding and 2 discrete states of T/R. • Sequential model shows that as more O2 binds, T state subunits gradually become R state subunits.

13. Protein Folding • Assisted by hsp70 proteins to protect exposed residues, chaperone proteins to provide hydrophobic capsule in which to fold • 6 steps • Nucleation—random coils to 2° structures • Condensation—association and growth of 2° structures • Molten globule—domains form, hydrophobic effect not complete • 3° D formation—N-C polypeptide forms completely • 4° D formation—Subunits assemble into a complex with H-bonds and disulfide bridges • Global energy minimum—Final, native conformation for enzymatic activity

14. Denaturation • Heat denaturation prevents enzymatic activity of normal proteins due to deformation—preventable by using ion bridges instead of uncharged polar/hydrophobic interactions • Chaotropic agents scramble protein ex. Urea, guanidiumHCl • Reducing agents break CS-SC disulfide bridges ex. β-mercaptoethanol, dithiothreitol (DTT) • Removing reducing agent before chaotropic agent forms scrambled intermediate with CS-SC bridges, useful to study intermediates of protein folding • Adding catalytic amount of reducing agent will restore native conformation