Ligand-Protein Interactions and Hemoglobin

• Ligands are atoms or molecules interacting with proteins through non-covalent interactions.
• Ligand binding is reversible.

Ligand-Protein Interactions

• Association reaction: L + P -> LxP
• Dissociation reaction: LxP -> L + P
• Stronger interaction results in a lower likelihood of separation.
• Stronger interaction implies higher affinity.
• Binding equilibrium yields a hyperbolic curve when % binding is plotted against ligand concentration.
• % binding increases as ligand concentration increases.
• Kd represents the ligand concentration at 50% binding.
• Lower Kd value indicates higher affinity.

Myoglobin and Hemoglobin

• Myoglobin and hemoglobin α and β subunits are evolutionarily related.
  • Evidenced by sequence and structural similarity.
• Myoglobin retains high O2 affinity in tissues.
• Hemoglobin is saturated in lungs and releases O2 in tissues.
• Hemoglobin rapidly responds to drops in O2 tension in tissues, shown by a sharp S curve.
• Hemoglobin cooperativity requires quaternary structure.
• Hemoglobin's binding curve is sigmoidal, not hyperbolic.
• Binding of the first ligand increases affinity for the next (positive cooperativity being common, negative cooperativity being rare).
• Myoglobin acts as an oxygen reservoir in muscles because it has a high affinity for oxygen.
• Hemoglobin has cooperative binding, and efficiently loads oxygen in the lungs and releases it in tissues.

Catalysts

• Catalysts increase the rate of a chemical process and often involve a chemical reaction.
• Catalysts can also transport ions across the membrane.
• Catalysts don't change the equilibrium or free energy change of a reaction.
• Many thermodynamically-favorable reactions are slow on biological timescales.
• Nearly every chemical process in living organisms is catalyzed as uncatalyzed reactions are too slow for biological timescales.
• Controlling enzyme activity regulates cellular processes.
• Proteins persist despite thermodynamically favorable hydrolysis into amino acids because they are not at equilibrium, requiring enzyme catalysts for breakdown.
• Protein breakdown via hydrolysis of peptide bonds is thermodynamically favorable.
• Protease catalyzes peptide bond hydrolysis to generate amino acids.
• An enzyme active site is a cleft or groove formed when an enzyme folds into its tertiary structure.
• The active site is not optimized for substrate binding.

Enzymatic Catalysis Equilibria

• E + S ⇌ ES ⇌ EP ⇌ E + P
  • Substrate binds to the active site.
  • Chemical reaction takes place in active site.
  • Product leaves the active site, regenerating the free enzyme.

Reaction Coordinate and Transition State

• The reaction coordinate represents the reaction progress.
• The transition state energy level represents the point of highest free energy.
• ΔG‡ is the activation free energy.
• ΔG is the change in free energy.

Reaction Rates

• G < 0: Indicates a reaction proceeds in the forward direction
• G > 0: Indicates a reaction proceeds in the reverse direction
• G = 0: Indicates a reaction is at equilibrium
• The reaction rate is inversely proportional to the height of the free energy activation barrier.
• Enzymes lower the activation free energy stabilizing the transition state in the active site.
• Enzymes preferentially bind the transition state.
• The transition state involves covalent bonds in the process of forming and breaking.
• Transition states are extremely short-lived and cannot be studied directly.
• The Michaelis-Menten equation relates substrate concentration to the reaction rate, resulting in a hyperbolic curve.
• Km is similar to Kd but for a substrate.
• Vmax is the maximal rate and it occurs when the enzyme is fully occupied with the substrate.
• Enzyme efficiency increases when Km decreases and Vmax increases.
• Km can be estimated using Vmax/2.
• A "rate-determining step" is the slowest step in a pathway which has the lowest Vmax.

Allosteric Regulation

• Involves a small molecule that is not the substrate.
• The molecule binds to a site other than the active site.
• Often seen in feedback inhibition of pathways.
• The allosteric ligand induces a structural change in the active site.
• Activity can either increase or decrease.
• It can occur in a monomeric enzyme differentiating it from cooperativity.

Regulation by Phosphorylation

• A phosphoryl group is transferred on to an amino acid side chain with -OH
• The reaction is reversible.
• A rapid response is allowed to changing conditions.
• Kinase catalyzes this by transferring a PO4 group.
• Phosphatase reverses this by removing a PO4 group.
• Phosphorylation changes the size and charge of the sidechain, resulting in structural changes at the active site.
• Can increase or decrease activity.

Competitive Inhibition

• A competitive inhibitor binds to the enzyme active site.
• It prevents the substrate from binding.
• It typically resembles the substrate.

Lipids

• Lipids are molecules with substantial nonpolar character with some having amphipathic character.
• Important classes

General Characteristics of Natural Fatty Acids

• Long chain carboxylic acids
• Typically contain 14 to 24 carbons
• Have an even number of carbons
• Can be split into two groups: saturated, with no double bonds and unsaturated, with one or more double bonds.
• A cis-double bond causes a kink or bend in the structure.
• More double bonds result in more curvature.
• The last carbon is called omega carbon.

Omega Fatty Acids

• Omega-3 and omega-6 must be acquired through diet
• Omega-3 fatty acids are important constituents of phospholipids, e.g. DHA is high in retina, brain and important for early brain development
• Omega-3s are precursors for signaling molecules called eicosanoids

Affect of temperature on melting point

• For saturated fatty acids, melting point increases with length because of stronger van der Waals interactions from increased surface area requiring more energy to overcome
• For unsaturated fatty acids, melting point decreases with increased number of double bonds because the double bonds introduce kinks, hindering tight packing and weakening IMF
• More unsaturated = fewer van der Waals interactions.

Glycerolipid Structures

• Monoacylglycerol- 1 fatty acid chain
• Diacylglycerol- 2 fatty acid chains
• Triacylglycerol-3 fatty acid chains
• Fatty acid salts form micelles in water
• Fatty acids and lipids containing a single fatty acid form micelles in water
• Free fatty acids are bound to albumin when transported in the circulation.
• Free fatty acids are detergent-like and can be deleterious to cells if not bound to proteins
• Albumin transports free fatty acids.

Triacylglycerols

• 3 fatty acid chains
• Glycerol
• Triacylglycerols form a separate phase in water, and are too wide to form micelles
• Not sufficiently polar to overcome the hydrophobic effect and are thermodynamically favorable
• Dietary triacylglycerols are hydrolyzed to free fatty acids in the small intestine to facilitate absorption
• They are absorbed and then remade in intestinal cells for distribution through the body
• TAG cannot be absorbed.
• Fats that are not needed for energy generation are stored as a large droplet in adipocytes
• Major storage depot for fats.

Glycerol-based Phospholipids

• 2 fatty acids esterified to glycerol
• 3rd hydroxyl of glycerol participates in a phosphodiester bond
• A variable R-OH group completes the head
• Called amphipathic molecules as they have a highly polar head and nonpolar tails.

Characteristics of Phospholipids

• Too wide to form spherical micelles and form bilayers instead
• Shape allows them to pack together in a planar assembly
• The thermodynamic driver is the hydrophobic effect
• Cells need to maintain optimal membrane fluidity
• They do this by modifying the ratio of unsaturated to saturated fatty acids in their membrane phospholipids
• More unsaturated = more fluid
• Fewer unsaturated = less fluid
• Fluidity increases with temperature

Cholesterol

• Regulates membrane fluidity by modulating interactions between fatty acid tails and makes membranes more rigid.
• Increases amount of noncovalent interactions in the hydrophobic core and is used to make bile salts.
• Bile salts emulsify dietary fats and promote their breakdown and absorption

Membrane Proteins

• Integral membrane proteins- these span the membrane:
• Peripheral membrane proteins- associate with the surface of the membrane:

Impermeability of Phospholipid Bilayers

• Glucose is highly H bonded to water
• They need to be broken to pass through the membrane, no H bonds are formed in the membrane interior
• Change in enthalpy is unfavorable
• Transporters creates a polar channel through the membrane and can be highly specific
• The pattern of polar and nonpolar side chains is inverted from that of soluble proteins

Carbohydrates

• Hydrates of carbon formula: Cn(H2O)n
• n=3-7 in biological systems and have many isomers with the same formula
• Highly polar and water-soluble

Aldoses vs Ketoses

• C1 and C2 groups are swapped
• C3-6 are the same
• Fructose is a ketone
• Glucose is an aldehyde
• Glucose C5-OH attacks C1 carbonyl: 6 atom ring
• Equilibrium strongly favors the cyclic form of C6 carbohydrates
• 99% cyclic and 1% linear at equilibrium
• Cyclic forms of carbohydrates consist of two anomers.

Anomers

• A anomer, C1-OH points down
• B anomer C1-OH points up
• anomers are formed due to rotation around the C1-C2 bond in linear form
• C1 of an a anomer and C4 of another monomer occurs with the loss of a water molecule and a(1->4) linkage

Glycosidic bonds

• Glucose: a(1->4) linkage, three a(1->4) polymers joined at a(1->6) branchpoints
• Glycogen and starch: a(1->4) linkage, both consist of a(1->4) polymers with occasional a(1->6) branches
• Cellulose: b(1->4) linkage
• Humans lack the necessary enzyme, cellulase, to break down the beta-1,4 glycosidic bonds in cellulose
• A,b (1->2) linkage
• Redox reactions involve the transfer of electrons.

Carbon compounds ranking

• Rank carbon compounds from most reduced to most oxidized +4 = most oxidized
• 4= most reduced
• Higher + more positive= more oxidized
• NAD+ = oxidized
• NADH= reduced

Reactions in metabolic sense

• Some are irreversible in a metabolic but not thermodynamic sense
• Generally, a reaction that exceeds a G value of -10 kj/mol is metabolically irreversible in the forward direction
• Cellular metabolite levels cannot change sufficiently to reverse direction
• Irreversible steps are often regulated, often committed steps

Carbohydrates Digestion

• Occurs in the first bite, small intestine, a dextrinase, catalyses hydrolysis of a 1-6 glycosidic bond.
• There are separate transporters for glucose and fructose
• Glucose is transported through the intestinal cell to the blood (two transporters: SGLT2 and GLUT2)
• Small amounts of fructose are metabolized in the intestinal cell

Glycogen

• From the circulation & transporter stimulated by increase in blood glucose and insulin after a meal and exercise
• In liver: supplies glucose when blood glucose levels drop
• Maintains a blood glucose concentration of ~3-7 mM
• Liver carries ~24 hr supply
• In muscle: important for endurance activities
• More efficient than uptake from blood and can be increased by training.
• Glycogen branching greatly increases the efficiency of glucose release.
• Isomerized to glucose-6-phosphate which enters glycolysis
• If glycogen is normal in structure and present in unusually large quantities in skeletal muscle tissue
• Symptoms:
• no problems with low intensity exercise
• rapid exhaustion of muscles on exertion
• severe muscle cramping
• breakdown of muscle cells
• no generation of lactate during exertion

Glycolysis

• Does not require oxygen (anaerobic) and takes 10 chemical steps
• generates 2 ATP per glucose
• inefficient in terms of energy extracted but fast
• short (1-2 mins) high intensity efforts can be powered by glycolysis alone
• longer duration activities require aerobic oxidation to fully oxidize the products of Glycolysis to glucose in Step 1 benefits the cell

Phosphorylation

• transfer of a phosphoryl group from a phosphoanhydride to an -OH group, generating a phosphoester
• traps glucose in the cell
• Phosphoester will be elevated to higher free energy through the oxidation of glucose and transferred back to ADP
• Step 1 involves direct transfer of a phosphoryl group from ATP to glucose.

Energy rankings

• Mixed anhydrides> phosphoanhydride> phosphoesters
• Recognize that the isomerization in step 2 requires ring opening
• Recognition that steps 1 and 3 are metabolically irreversible and Steps 2,4,5 are reversible

Steps in Glycolysis

• First committed step commits fructose-6-phosphate to glycolysis. The enzyme that catalyzes this reaction is called PFK the rate determining step in Glycolysis with highly regulated allosteric activation and inhibition, - ring opens up and isomerization of aldose to ketose occurs