MCB 150 61-90 Lecture Notes: Proteins, Enzymes, and Cellular Respiration

Exam 1 Information

• Exam 1 is next week, from Thursday to Sunday, at the CBTF.
• The exam consists of 35 questions, each worth 4 points, with a second chance option for 1 point.
• Monday’s lecture content will be included in Exam 1.
• Wednesday’s lecture marks the beginning of Unit 2 material, which will not be on Exam 1.
• There will be no class or student hours on Friday.

Protein Structure and Function

Secondary Structure

• Hair protein (keratin) is rich in α-helical structures.
• Hair stretches easily because the hydrogen bonds stabilizing α-helices are easily broken.

Prions

• Prions are misfolded proteins that induce normal versions of the same protein to fold incorrectly.
• Misfolded proteins come out of solution and create plaques.
• Prions cause diseases called spongiform encephalopathies, such as Mad Cow disease, Scrapies, and Creutzfeldt-Jakob disease.

Tertiary Structure (3°)

• Tertiary structure refers to the unique 3D folded structure of a protein.
• It represents the final conformation of some proteins.
• It is due to interactions between R-groups with each other and with the backbone.
• Stabilizing factors include:
  • Hydrogen bonds between polar or charged side chains.
  • Hydrogen bonds between hydrophilic side chains and the backbone.
  • Ionic bonds between acidic and basic amino acids.
  • Hydrophobic clustering of non-polar side chains.
  • Van der Waals forces.
  • Disulfide linkages.
• Water molecules surround the protein, causing it to contort so that hydrophilic R groups are on the outside and hydrophobic R groups are on the inside.
• It lines up sites for functional activity of that protein.

Quaternary Structure (4°)

• Quaternary structure is found in proteins with multiple polypeptide chains (subunits).
• Subunits can be the same or different:
  • 2 identical subunits = homodimer
  • 2 different subunits = heterodimer
• Ferritin (iron storage protein) has 24 identical subunits.
• Hemoglobin (Hb) consists of 4 separate polypeptides (2 α and 2 β chains).
  • The sickle cell mutation changes a glutamic acid(Glu, hydrophilic) to valine (Val, hydrophobic).
  • The affected amino acid is on the outside of the protein.
  • Hb molecules stick together to “hide” Val from water.
  • When oxygen levels fall, Hb precipitates and distorts red blood cells (RBCs).

Relative stabilities of biomolecular forces:

• Disulfide linkages → Covalent
• Ionic bonds → Easily made and broken
• Hydrogen bonds
• Hydrophobic clusters
• Van der Waals forces

Protein Folding and Denaturation

• The information for how a protein will fold resides in its primary structure.
• Removal or inactivation of stabilizing forces unfolds (denatures) the protein to its 1° structure, without breaking peptide bonds.
• Denaturation leads to the loss of 2° and 3° structure, almost always resulting in loss of function.
• Denaturing agents include acids, bases, heat, and detergents.
• If the denaturing agent is removed, some proteins will resume their properly folded 3D structure, indicating that the “instructions” are in the 1° structure.

Enzymes and Biological Catalysis

• Many proteins are enzymes: biological catalysts that facilitate biological reactions.
• This is necessary because most cellular reactions proceed at a very slow rate without them.
• Two broad categories of cellular reactions based on change in energy level (E):
  • Reactions that require an input of energy.
  • Reactions that release energy upon completion.

Anabolic vs. Catabolic Reactions

• Reactions that require energy are called biosynthetic or anabolic. They involve linking together smaller molecules into larger ones (e.g., condensation reactions of monomers to macromolecules).
• Reactions that release energy are called catabolic. They involve breaking down larger molecules into smaller ones (e.g., hydrolysis reactions of macromolecules to monomers). They are also referred to as spontaneous reactions.

Spontaneity and Energy of Activation

• Two different meanings for the word spontaneous:
  • Typical meaning: happens automatically.
  • Biology meaning: a reaction that releases energy, much of which is lost as heat.
• Catabolic (energy-releasing) reactions require a certain amount of energy to get started (Energy of Activation, or E_a).
• The energy could come from heat, but enzymes are used instead.

Standard Activation Energy Diagram

• [S] = energy level of substrate (reactants).
• [P] = energy level of products.
• E_a = activation energy, which converts substrates into unstable transition states.
• \Delta G = Free Energy of Reaction: difference in E between reactants & products.

Enzymes and Reaction Rates

• Enzymes do not cause reactions to occur that would not eventually occur anyway; they only speed up existing reactions.
• Many enzymes increase reaction rates by several million times; some by several trillion times.
• Example:
  • 2H2O2 \leftrightarrow 2H2O + O2
    • Platinum (inorganic catalyst) decreases E_a by 1/3rd.
    • Catalase (enzyme) decreases E_a by almost 90%!

Enzyme Specificity and Active Sites

• Enzymes bind substrates with extremely high specificity into their active sites (usually just a few amino acids).
• Enzymes will most likely cause some conformational change in the substrate molecule(s), but they themselves usually change shape upon binding substrate (induced fit).

Mechanism of Enzyme Action

• How does substrate binding to the active site decrease E_a?:
  • Acting as a template for substrate orientation.
  • Stressing the substrate(s) and stabilizing the transition state.
  • Providing a favorable microenvironment.
  • Participating directly in the catalytic reaction.
• If an enzyme accepts a group from a substrate, it must in turn donate that group to help form a product.
• Enzymes are (ultimately) unchanged by the reactions they catalyze.
• Enzymes do not change the equilibrium of reactions; they only make it easier (and faster) to reach that equilibrium.
• Enzymes decrease E_a by the same amount in both directions.

Factors Affecting Enzyme Activity

• Because most enzymes are proteins, conditions that affect protein stability also affect enzyme activity.
• Enzymes have temperature and pH optimums.
• Most tend to be near body temperature (37 °C) and neutral pH (7.0).

Enzyme Inhibition

• Enzyme + Substrate -->> [Enzyme-Substrate Complex] -->> Enzyme + Product
• Enzyme + Inhibitor -->> [Enzyme-Inhibitor Complex] (No Product Formed)
• Inhibition can be either reversible or irreversible.
• Reversible inhibition can be competitive or noncompetitive.

Irreversible Inhibitors

• Permanently bind to or modify the active site; changing the concentration of natural substrate or inhibitor has no effect.
• Nerve agents like sarin gas are irreversible inhibitors of acetylcholinesterase, which catalyzes the termination of nerve impulses.
• Tend to be molecules not typically encountered by that particular cell.
• Irreversible inhibition demonstrates that enzymes must ultimately be unchanged if they are to be used over and over.

Competitive Inhibition

• The inhibitor molecule physically resembles the natural substrate and occupies the active site.
• The enzyme can’t use the inhibitor as a substrate; no products are formed.
• Can be “flooded out” by increasing the concentration of the natural substrate.
• Decreasing the concentration of the inhibitor also reduces the probability of the inhibitor finding the active site.
• Example: In bacteria (but not humans), DHPS catalyzes the conversion of p-aminobenzoic acid into folic acid; sulfa drugs like sulfanilamide are inhibitors of DHPS; bacteria die, but humans are unaffected.

Noncompetitive Inhibition

• The inhibitor molecule binds to the enzyme in a place other than the active site.
• If the change in the enzyme completely prevents substrate binding, increasing the substrate concentration has no effect.
• Reversible because the inhibitor can become unbound.

Enzyme Biochemistry

• V{max} and KM
• Effect of Competitive and Noncompetitive Inhibitors on V{max} and KM

Cellular Respiration

• We eat food to give us energy, but how does the energy from food get to ATP?
• Cellular Respiration: The breakdown of glucose to CO2 and H2O
• Multiple reactions in 3 distinct pathways or “phases”:
  • Glycolysis
  • Pyruvate oxidation and Krebs cycle
  • Oxidative phosphorylation (electron transport and chemiosmosis)

Phase 1: Glycolysis

• “Glyco” (sugar) + “lysis” (splitting)
• Starts with a 6-carbon sugar (glucose), ends with two 3-carbon molecules (pyruvate).
• Pathway is actually endergonic up to the production of first 3-carbon molecules (uses the cell’s store of ATP).
• Occurs in the cytoplasm of all living cells.
• In glycolysis:
  • 2 steps are endergonic
  • 3 steps are exergonic
  • This energy is harnessed and saved for later
  • 2 Substrate-level phosphorylation reactions

Problems at the end of glycolysis:

1. Molecules still are not at their lowest energy state
2. Some of our energy is being held in NADH
3. NAD^+ is being used up and not replaced

Leads to new questions:

1. How do we get more energy out of pyruvate?
2. How do we transfer the energy in NADH to ATP?
3. How do we regenerate NAD^+?

Answer to previous questions:

• It depends on the presence or absence of oxygen (O_2) or other terminal electron acceptor
  • If oxygen (O_2) is present, cells will undergo aerobic respiration.
  • If oxygen (O_2) is absent but an alternative terminal electron acceptor exists, cells will undergo anaerobic respiration.
  • If oxygen (O_2) is absent and no terminal electron acceptor exists, cells might be able to undergo fermentation.

Aerobic Respiration:

• Carbon source (2 molecules of pyruvate) completely converted to carbon dioxide
  • Pyruvate molecules first converted to acetyl-CoA, which then enters the Krebs (or Citric Acid) Cycle
  • All C-H bonds converted to C-O bonds (6 CO_2 released)
• More energy transferred to NAD^+ and FAD (makes more NADH & FADH_2)
• Another SLP reaction in Krebs cycle (GTP is ATP analog)
• Occurs in mitochondria of eukaryotes; cytoplasm and plasma membrane of prokaryotes

Mitochondria:

Organization of mitochondria:

• Inner Membrane
  • Principal site of ATP generation
  • >70% protein (no porins)
  • Impenetrable to ions & small molecules except by transporters
• Outer Membrane
  • Typical protein %
  • Porins
• Intermembrane Space (IMS)
  • Composition of ions and small molecules is the same as the cytoplasm
• Matrix
  • Krebs enzymes
  • DNA & ribosomes

Phase 2: Pyruvate Oxidation and Krebs Cycle

Net results of Phase 1 (Glycolysis) and Phase 2 (Pyruvate Oxidation and Krebs Cycle) for every glucose:

Problems at the end of Krebs Cycle:

1. Still haven’t replaced NAD^+; in fact, more NADH is made
2. Now you have FADH_2 that needs to be re-oxidized
3. Still haven’t transferred energy carried by cofactors to ATP

• Also, why is this dependent on oxygen?
  • Aerobic respiration requires oxygen, but Krebs Cycle itself does not
  • Because Krebs Cycle is coupled to the next pathway which does require oxygen –– the Electron Transport Chain

Oxidative Phosphorylation:

The Electron Transport Chain:

• NADH passes its electrons (and is re-oxidized to NAD^+​) to the first carrier in the membrane
  • This ends NAD^+​ / NADH's involvement, and NAD^+​ is now free to participate in another redox reaction
• First electron carrier passes to second, second carrier passes to third, and so on
  • Because carriers are at successively lower energy levels, energy is released when the electrons are passed
  • This energy is used to pump protons across the membrane
    • a proton gradient (aka electrochemical gradient) is produced
• Last electron carrier passes electrons to oxygen, which combines with protons to form water
  • Now we’ve accounted for the CO2 (Krebs) and H_2O (ETC)
• FADH_2 also joins the party, but passes its electrons to a carrier down the line
  • Bypasses Complex I
  • Not as many protons pumped across the membrane
    • Less of a contribution to the overall electrochemical gradient
• Regenerated cofactors and built gradient, but no ATP!