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• Hemoglobin (Hb)


  • Provides tissues with a continuous O2 supply
  • Transports O2 from the lungs or gills to the respiring tissues for aerobic metabolism in the mitochondria

  
• Myoglobin (Mb)


  • Aids transport of O2 to the mitochondria
  • Can store O2
• Oxyhemoglobin


  Hb when O2 is bound
• Deoxyhemoglobin


  Hb without O2 bound
• Myoglobin and Hemoglobin


  • Bind to iron Fe2+
  • Have tertiary and quaternary structure
• The relatively high concentration of myoglobin (2mg/g human muscle) facilitates oxygen diffusion in cells so it reaches mitochondria efficiently
• In deep sea diving animals concentration myoglobin in skeletal muscle is 30 X greater than terrestrial animals
• Heme


  • Porphyrin = tetrapyrrole ring
  • Mb or Hb without heme = apoprotein
  • Mb or Hb with heme =holoprotein
• Proximal histidine residue


  Of the globin protein
• Distal His


  Stabilizes the bound O2 via H-bonding
• Oxygen binding curve


  • Hyperbolic
  • Sigmoidal
• P50


  • A measure of O2 binding affinity
  • P50 is pO2 at half saturation
  • Globin with a higher O2- binding affinity, the value of P50 is lower
  • Globin with a lower O2- binding affinity, the value of P50 is higher
• O2 binding to myoglobin

  • Mb binds and releases O2 depending on the O2 concentration in the cell
  • Mb releases its O2 supply to the mitochondria for production of ATP
  • Mb serves as a buffer of intracellular oxygen concentrations, keeps O2 concentration steady and acts as an O2 reservoir in muscle
  • Mb has a greater affinity for O2 at all partial pressures of O2 (than Hb)
• Cooperativity


  • Hb changes it's conformation to increase or decrease its affinity for O2 depending on how much O2 is around = a perfect transport protein
  • Cooperativity in binding requires communication between binding sites
• Allosteric effect


  • Cooperative binding of O2 by Hb - an allosteric effect
  • Allosteric binding - the uptake of one ligand by a protein influences the affinities of remaining unfilled binding sites
  • Ligands - same (binding to Hb), or different
  • Allostery - regulating the activity of enzymes
  • Allosteric interactions - when an allosteric effector/ modulator binds to a protein and changes the proteins activity
  • Allosteric effector - O2; it binds to Hb and changes its activity - it makes Hb want to bind to more O2 molecules
  • Allosteric protein – Hb, a protein whose activity is changed by an allosteric effector
• T and R state

  • Hemoglobin adopts the T state or the R-state conformation depending on the oxygen concentration in the environment
  • There is a 15˚ rotation of α1β1 with respect to α2β2 upon switching from the T to R state
  • Narrowing of central channel during the T → R transition
  • T-state Hb has a lower O2 binding affinity (Higher P50)
  • R-state Hb has a higher O2 binding affinity (Lower P50)

  
• Positive homotropic effector


  Bind at binding or active site; O2; binding ↑ binding affinity of O2 to other hemes in the tetramer
• Negative heterotropic effectors


  • Bind at other sites on Hb and cause allosteric effect
  • H+, CO2, and 2,3-bisphosphoglycerate (2,3-BPG)
  • ↓ the binding affinity of O2 to Hb
• 2,3-BPG


  • Inside red blood cells
  • Potent allosteric effector
  • Lowers O2 affinity of Hb
  • Stabilize the T state
  • Promote greater O2 delivery/release to tissues
• Carbamate


  1. 13% CO2 is bound to Hb amino groups
• Llama vs fetal hemoglobin

  • Hb with a higher affinity for O2 (lower P50)
  • Reduced heart rate and a lower metabolic rate, larger lung capacity
  • Increased Mb concentration
  • Increased BPG
  • β chain is replaced with a γ chain
  • Fetal α2γ2 Hb does not bind 2, 3-BPG as well as adult Hb does
  • A higher affinity for O2, binding O2 when the mother's Hb is releasing O2
• Bohr effect


  • pH effect on O2 transport
  • Stimulation of O2 release from Hb by CO2 and H+
  • Accumulation of CO2 lowers the pH in erythrocytes through the bicarbonate reaction catalyzed by carbonic anhydrase
  • A decrease in blood pH - stabilization of the deoxy T state and greater O2 released from Hb
  • At lower pH, salt bridges form that stabilize the T state
• Sickle-cell anemia


  • A genetic disease caused by Hb mutation resulting in the change of Glutamate to Valine at position 6 in the two Hb β chains
  • Affects 0.4 % of Afro-American
  • The β6 mutation (Glu to Val 6) = a protrusion from the circle in the β2 subunit
  • The hydrophobic pocket containing Phe85 and Leu88 = a nick in the β1 subunit
  • Interaction of different Hb S tetramers to form the fibres
  • The tetramers are in the deoxy state to form the fibres to form
• Enzymes


  • Proteins that catalyse almost all reactions in the living cells
  • Speeding up a chemical reaction
  • High specificity - affinity can be 1000x greater than closely related compounds; varying degrees of specificity
  • 'Green' catalysts (natural, non-toxic & biodegradable) with high (chiral & structural) selectivity coupled with efficiency (speed)
  • Enzyme reactions - main targets for medicinal agents
  • Regulation / activity is regulated
  • Cofactor - small inorganic/metal ions (Cu, Mg, Mn, Fe), activators &/or inhibitors
  • Coenzyme - small organic non-protein ligand that catalyze reactions…+/- electrons, transfer a group, form or break a covalent bond (NAD+, CoA, vitamins)
  • Prosthetic group - large complex organic molecules, which may have catalytic activity (e.g. heme in hemoglobin) – covalently bound!
  • Active site - portion of E which folds to precisely fit the contours of a S via weak electrostatic interactions & facilitates bond reactivity
  • Enzyme-substrate (ES) complex - unique joining of E & S at active site
• Enzyme classes


  • Enzyme names often end in –ase and their name describes their function
• Enzymes


  • Increase the velocity of a reaction by accelerating the approach to equilibrium
  • Change rates of processes but do not affect the position of equilibrium
  • Lower the free energy of the transition state for the reaction they catalyse
  • Don't change the thermodynamic favorability of a reaction
• Enzyme reaction path


  • E + S <---> [ES] <---> E + P
  • Enzymes catalyze reactions by lowering the energy of activation... Ea
  • There is no difference in free energy between an enzyme catalyzed reaction and an un-catalyzed reaction, but an un-catalyzed reaction requires a higher energy input than a catalyzed reaction!
• How enzymes act as catalysts


  • Enzyme active site complementary: shape, charge, polarity
  • But MOSTLY to transition state, not reactant
  • ES complementarity is the basis for the specificity
  • 2 reactants are bound to sites on the catalyst - ensures their correct mutual orientation and proximity
  • Binds them most strongly when they are in the transition state conformation
• Lock and Key model

  • Substrate binds to active site
  • Reaction occurs
  • Products desorbed
  • Leaving site open for new substrate molecule
  • Both E and S distorted on binding
  • S forced into a conformation approximating the transition state
  • Only the proper S can induce the proper alignment of the active site = some compounds can bind but not react!!!
• Induced Fit Theory


  Glucose binding to hexokinase induces the enzyme to fit around it
• Mechanisms for achieving rate acceleration

  • For many enzyme-catalyzed reactions the first step, binding of substrate, is reversible (i.e., k1 and k-1 >> k2)
  • The second step, conversion of ES to EP, lies far to the right (i.e., k2 >> k-2)
  • The third step, release of product, is rapid compared to the catalytic step (i.e., k3 >> k2)
• Transition state and tetrahedral intermediate for an enzyme-catalyzed ester cleavage

  • Enthalpic stabilization of transition state
  • General acid/base
  • Electrostatic stabilization
• Lysozyme mechanism


  • Glu35 acts as a general acid to promote cleavage of the glycosidic bond and formation of the oxocarbenium ion stabilized electrostatically by Asp52
  • Glu35 acts as a general base, deprotonating a water molecule, which then attacks C1 of the substrate
  • Formation of covalent intermediate
  • Glu35 protonated (general acid) pH below 6.2
  • Asp52 deprotonated to interact with the oxocarbenium ion, pH above 3.7
  • pH optimum ~5 Glu35 is protonated and Asp52 deprotonated
• Chymotrypsin


  Serine protease
• Michaelis-Menten kinetics


  • Michaelis-Menten equation - one of the best-known models of enzyme kinetics; describes how the chemical rate catalyzed by an E depends on [S], the turnover rate, and how tightly the E binds its S
  • KM is a binding constant of the 'affinity of a substrate for the enzyme'
  • kcat is a measure of catalytic efficiency; how fast can E convert S to a P: kcat = turnover number = [moles of S turned over] per [mole of E] per second
  • The ratio kcat/KM is a measure of how good an E is – E would be 'perfect' if it catalysed reactions at the diffusion limit
• Steady state in enzyme kinetics

  • [E]t = [E] + [ES]
  • The steady state assumption proposes that the concentration of E-S complex remains nearly constant through much of the reaction
  • We can therefore calculate the reaction velocity by assuming steady state conditions!
• Enzymes display saturation kinetics


  • Reaction velocity as a function of substrate concentration
  • Under the steady-state assumption, the concentration of E-S complex remains nearly constant through much of the reaction
  • The Michaelis constant, KM, indicates the [S] at which the reaction rate is ½ Vmax
  • The turnover number, kcat, measures the rate of the catalytic process
• Lineweaver–Burk plot


  • In this double reciprocal plot, 1/v is graphed versus 1/[S]
  • Linear extrapolation of the data gives both Vmax and KM
  • Km reflects how well substrate binds to enzymes, smaller Km means better substrate binding
  • k cat reflects rate at which enzyme can go through reaction mechanism (kcat = Vmax / Et)
• Order of substrate binding

  • Random S Binding
  • Ordered S Binding
  • The Ping-Pong Mechanism
• Enzyme inhibition


  • A decrease in the catalytic activity as consequence of the change of reaction conditions (e.g., temperature, pH, [S] or [P])
  • These conditions can cause conformational changes or blocking of the enzyme active site
  • Inhibitor = a substance that decreases the rate of an enzyme-catalysed reaction when it is present in the reaction mixture
  • Ireversible – covalently bound; I can not be easily removed from E; some antibiotic drugs, such as penicillin form covalent link to active site
  • Reversible – non-covalently bound; E activity may be restored by removing the I
  • Complete inhibition (linear)
  • Partial inhibition (hyperbolic)
  • Competitive inhibition - I and S compete for E
  • Non-competitive inhibition - I can bind to E at the same time as the S
  • Uncompetitive inhibition - I can not bind to the free E, but only to the ES-complex
  • Mixed inhibition -This type of inhibition resembles the non-competitive, except that the EIS-complex has residual enzymatic activity