Enzymes

Enzyme structure and function

• Function of enzymes in catalyzing biological reactions
  • Enzymes are catalysts, which are things that increase the rate of a reaction, but does not get used up during the reaction.
  • Structure determines function. A change in structure => a change in function.
  • Important biological reactions catalyzed by enzymes:
    • Metabolism
    • DNA synthesis
    • RNA synthesis
    • Protein synthesis
    • Digestion
• Enzyme classification by reaction type
  • Enzyme class = reaction type + "ase"
  • Oxidase, reductase = oxidation, reduction reaction
  • Dehydrogenase = removes hydrogen (oxidation), can also work in reverse (reduction)
  • Kinase (adds), phosphatase (removes) phosphate groups
  • Protease, amylase, lipase = hydrolyzes proteins, carbohydrates, fats
  • DNAse, RNAse, nuclease = hydrolyzes phosphodiester bonds in nucleic acids
  • Polymerase = makes DNA/RNA/protein (polymers of nucleic acids/amino acids)
  • Transcriptase = transcribes RNA from DNA template (reverse transcriptase makes DNA from RNA template)
  • Ligase = forms bonds between 2 strands of DNA/RNA
• Reduction of activation energy
  • 
  • Enzymes decrease the activation energy (E_a) of a reaction by lowering the energy of the transition state.
  • Enzymes increase the rate of a reaction by decreasing the activation energy.
  • Enzymes will increase the rate constant, k, for the equation rate = k[A][B].
  • Enzymes do NOT change the K_eq of a reaction.
  • Enzymes do not change K_eq because it lowers the activation energy for BOTH forward and reverse reactions.
  • Enzymes will make the reverse reaction go faster also.
  • Enzymes do not change ΔG, the net change in free energy.
  • Enzymes affect the kinetics of a reaction, but not the thermodynamics.
• Substrates and enzyme specificity
  • Enzyme-substrate interactions occur at the enzyme's active site.
  • Enzyme-substrate specificity derives from structural interactions.
  • Lock and key model: rigid active site. Substrate fits inside the rigid active site like a key.
  • Induced fit model: flexible active site. Substrate fits inside the flexible active site, which is then induced to "grasp" the substrate in a better fit.
  • Enzymes can be specific enough to distinguish between stereoisomers.
• Mechanisms of catalysis
  • Enzymes can be protein or RNA.
    • Almost all enzymes in your body is made of protein.
    • The most important RNA enzyme in your body is the ribosome.
  • Structure determines function; Enzyme structure derives from 4 levels.
    • Primary: this is the sequence of the protein or RNA chain.
    • Secondary: this is hydrogen bonding between the protein backbone. Examples include alpha helices and beta sheets (backbone H-bonding). For RNA, this is base pairing.
    • Tertiary: this is the 3-D structure of the enzyme. This involves -R group interactions and spatial arrangement of secondary structure.
    • Quaternary: when more than 1 chain is involved. When you hear about "dimers", "trimers", "tetramers", "oligomers", that's quaternary structure.
  • Cofactors = metal ions (DNA polymerase needs magnesium)
  • Coenzymes = small molecules (NAD, FAD, CoA, vitamins)
  • Fat soluble vitamins: Vit A, D, E, K. Can't be excreted in urine, so can be toxic at high levels
    • A: night vision (night blindness if deficient)
    • D: bones (Rickets if deficient in kids, Osteomalacia in adults)
    • E: cell membranes (deficiency causes hemolytic anemia, neuropathy, myopathy)
    • K: clotting (Bleed if deficient. Warfarin blocks vitamin K pathway and works as a blood thinner. Premature infants lack vitamin K because they haven't had time to acquire the gut bacteria that makes vitamin K)
  • Water soluble vitamins: anything that's not fat soluble. Less toxic as it can be peed out
    • B1/thiamin: deficient in alcoholics (Wernicke-Korsakoff syndrome) or dietary deficiency (Beriberi)
    • B12/cobalamine: needed in DNA synthesis. Acquired through diet. Deficient in strict vegetarians or inflammatory bowel disease. Causes macrocytic anemia and neuropathy
    • Folate: needed in DNA synthesis. Deficient in alcoholics or dietary lack of vegetables. Causes macrocytic anemia. Anti-folate drugs block folate pathways to kill rapidly dividing (DNA synthesizing) cancer cells.
    • Vitamin C: needed in collagen synthesis. Deficiency causes scurvy
  • Effects of local conditions on enzyme activity
    • Enzymes have optimal conditions (temperature, pH, salt concentration) where they function the best
    • Usually this is body temperature, but the immune system functions better at 1 degrees higher. This is why you get a fever when you get an infection.
    • Too much fever can denature enzymes and cause permanent damage. This is why antipyretic drugs (acetaminophen) are used to treat fever.

Control of enzyme activity

• Kinetics
  • General (catalysis)
    • Enzyme "catalyzes" a reaction, meaning it changes the kinetics of a reaction without being used up
    • Kinetics (how fast a reaction occurs) vs Thermodynamics (whether and what direction a reaction will occur)
    • Enzyme makes a reaction go faster by lowering the activation energy
    • Enzymes lower the activation energy by stablizing the transition state (high energy state between reactant and product)
  • Michaelis-Menten = reaction rate (V) vs substrate concentration [S] plot
    • Vmax = maximum reaction rate = V where [S] is infinity
    • Km = Michaelis constant = [S] where V is half of Vmax
  • Cooperativity
    • Positive cooperativity = binding of substrate from one subunit makes another subunit more likely to bind
    • Negative cooperativity = binding of substrate from one subunit makes another subunit less likely to bind
• Feedback regulation
  • The product of a pathway inhibits the pathway.
  • For example, hexokinase, the first enzyme in glycolysis, is inhibited by its product glucose-6-phosphate.
• Inhibition - types
  • Competitive inhibition
    • 
    • 
    • An inhibitor competes with the substrate for binding to the active site.
    • Competitive inhibition increases Km (the amount of substrate needed to achieve maximum rate of catalysis).
    • Competitive inhibition does NOT change Vmax (the maximum possible rate of the enzyme's catalysis).
    • Michaelis Menten plot: less hyperbolic curve
    • Lineweaver Burk plot: counterclockwise rotation around the y intercept
    • You can overcome competitive inhibition by providing more substrate.
  • Non-competitive inhibition
    • 
    • 
    • An inhibitor binds to an allosteric site on the enzyme to deactivate it.
    • The substrate still have access the active site, but the enzyme is no longer able to catalyze the reaction as long as the inhibitor remains bound.
    • Non-competitive inhibition decreases Vmax (the maximum possible rate of the enzyme's catalysis).
    • Non-competitive inhibition does NOT change Km (the amount of substrate needed to achieve the maximum rate of catalysis).
    • Michaelis Menten plot: lower plateau
    • Lineweaver Burk: counterclockwise rotation around the x intercept
    • You can't overcome non-competitive inhibition by adding more substrate.
  • Mixed
    • Michaelis Menten plot: lower plateau AND less hyperbolic curve
    • Lineweaver Burk plot: both y and x intercept changes (y intercept shifts up, x intercept shifts right)
  • Uncompetitive inhibition
    • 
    • 
    • Inhibitor binds the enzyme-substrate complex
    • Michaelis Menten plot: lower plateau but more hyperbolic curve
    • Lineweaver Burk plot: parallel shift upward
• Regulatory enzymes
  • Allosteric enzymes: binding at allosteric site -> change in enzyme conformation -> change in active site to bind substrate more easily
  • Covalently-modified enzymes: phosphorylation of an enzyme makes it active/inactive
  • Zymogen: inactive until activated by another enzyme. Eg: Pepsinogen (inactive) -> pepsin (active); trypsinogen (inactive) -> trypsin (active)