Phu Review 3

Phu Review 3 - BIBC 100 Final Review Session (Final...

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Unformatted text preview: BIBC 100 Final Review Session (Final Saturday 8­10:50am) Enzymes Enzymes ► Biological catalysts that lower the activation energy of a reaction Favors/stabilizes intermediate formation over substrate Michaelis­Menten 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]) Lineweaver­Burk plot is a reciprocal of MM equation, using straight lines instead of hyperbola 1/vo = (Km/Vmax) x (1/[S]) + 1/Vmax Serine Protease 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 Serine Protease 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, peptide­N­terminal 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 peptide­C­terminal and reformation of Serine hydroxyl using H+ from His, peptide­C­terminal half leaves Membrane Transporters Membrane Transporters ► Channels facilitate diffusion (ex. KcsA K+ channel) ► Pumps require energy (ex. SERCA pump) Up/against concentration gradient Requires energy, ex. ATP Down/with concentration gradient Does not require energy to transport Similar to enzymes in that they lower energy cost to cross the membrane ► ► Transmembrane domains, identifiable with hydropathy plot (hydropathy index >1 at consecutive transmembrane residues in α­helices) Nicotinic acetylcholine receptor (nAChR) and bacterial KcsA K+ channel nAChR nAChR ► Ligand­gated cation channel in skeletal muscle cells at the neuromuscular junction (NMJ), 5x 4­α­ 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 KcsA K Channel KcsA K + ► ► ► ► ► Bacterial Potassium membrane channel, 4x 3­α­helical 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=O­­­K+ 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 Calmodulin Calmodulin ► ► ► Calcium­modulated protein (CaM) with 4x EF­hand motifs (2x α­ helices) that bind up to 4 Ca2+ ions Linker α­helix 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 ► Ca2+/CaM­activated Ca2+ pumps on plasma membrane and sarcoplasmic reticulum keep intracellular/cytosolic [Ca2+] low to allow muscle contraction/relaxation cycles and calcium waves 4Ca2+/CaM­MLCK – Activates MLCK which (+P) MLC which allows crossbridge formation ­> contraction in smooth muscles 2Ca2+/CaM­K+ ­ CaM­activated K+ channels allows repolarization of depolarized membrane of neuronal/muscular cells 2Ca2+/CaM­EF – CaM­activated 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 Myoglobin 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 h­bond 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 Hemoglobin 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) Allosteric regulation Gives up O2 to myoglobin at tissues Gives up O2 to fetal Hb, which has higher aff. ► 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 Cooperativity 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 α1­β2 and α2­β1 h­bonds 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. ► Protein Folding 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—N­C polypeptide forms completely 4° D formation—Subunits assemble into a complex with H­bonds and disulfide bridges Global energy minimum—Final, native conformation for enzymatic activity Denaturation 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 CS­SC disulfide bridges ex. β­ mercaptoethanol, dithiothreitol (DTT) Removing reducing agent before chaotropic agent forms scrambled intermediate with CS­SC bridges, useful to study intermediates of protein folding Adding catalytic amount of reducing agent will restore native conformation ...
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