Molecular Biology: Enzymes and Metabolism

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
  • Reduction of activation energy
    • reaction diagram catalyzed reaction
    • Enzymes decrease the activation energy (Ea) 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 Keq of a reaction.
    • Enzymes do not change Keq 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.
    • 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.
    • 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.
    • Heat and extreme pH denatures enzymes by altering their structure.

Control of enzyme activity

  • Feedback inhibition
    • The product of a pathway inhibits the pathway.
    • For example, hexokinase, the first enzyme in glycolysis, is inhibited by its product glucose-6-phosphate.
  • Competitive inhibition
    • An inhibitor competes with the substrate for binding to the active site.
    • Competitive inhibition increases the amount of substrate needed to achieve maximum rate of catalysis.
    • Competitive inhibition does NOT change the maximum possible rate of the enzyme's catalysis.
    • 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 the maximum possible rate of the enzyme's catalysis.
    • Non-competitive inhibition does NOT change the amount of substrate needed to achieve the maximum rate of catalysis.
    • You can't overcome non-competitive inhibition by adding more substrate.

Basic metabolism

  • Metabolism consists of two parts: Catabolism and anabolism.
  • Catabolism is breaking stuff down for energy. This is the part that the MCAT (and what we) focuses on.
  • Anabolism is using energy to build stuff for storage.
  • Unless otherwise stated, everything here on metabolism is about catabolism - breaking things down for energy.
  • Another name for metabolism is cellular respiration.
  • Steps of aerobic metabolism (needs oxygen)
    • Glycolysis
    • Oxidative decarboxylation
    • Krebs cycle
    • Electron transport chain.
  • Steps of anaerobic metabolism (don't need oxygen)
    • Glycolysis
    • Alcohol or lactic acid fermentation
  • Aerobic metabolism of glucose
    • Complete oxidation of metabolite (glucose) to carbon dioxide.
    • ~30 ATP produced per glucose.
    • C6H12O6 + 6O2 => 6CO2 + 6H2O
    • C6H12O6: this is glucose. You get it from your diet.
    • 6O2: this is molecular oxygen that you breathe in.
    • 6CO2: this is carbon dioxide produced by the Krebs cycle. Both the carbon and oxygen in this CO2 comes from the metabolite (glucose).
    • 6H2O: this is water produced in the electron transport chain. The oxygen comes completely from the molecular oxygen that you breathe in.
    • If we were to follow the carbon in the metabolite (glucose), it will end up in carbon dioxide.
    • If we were to follow the oxygen in the metabolite (glucose), it will end up in carbon dioxide.
    • If we were to follow the oxygen you breathe in, it will end up in water.
    • As for the hydrogens, they'll either be in water, exist as protons in solution, or be transferred to some other entity.
    • As we can see, the total reaction involves complete oxidation of the metabolite (glucose) and complete reduction of molecular oxygen.
    • When electrons pass from the metabolite (glucose) to molecular oxygen, energy is released.
    • The electron transport chain harnesses this energy.
  • Anaerobic metabolism of glucose
    • Partial oxidation of metabolite (glucose) to pyruvate.
    • 2 net ATP produced per glucose.
    • Pyruvate is then reduced to either alcohol or lactate.
    • Bacteria reduce pyruvate to alcohol in a process called alcohol fermentation.
    • Humans reduce pyruvate to lactate in a process called lactic acid fermentation.
  • Glycolysis, anaerobic and aerobic, substrates and products
    • Glycolysis = convert glucose (6 carbons) to 2 molecules of pyruvate (3 carbons).
      • Location: cytosol.
      • 2 net ATP made for every glucose (2 input ATP, 4 output ATP).
      • 2 NADH made for every glucose.
      • Occurs under both aerobic and anaerobic conditions.
      • Glycolysis is inhibited by ATP.
    • Aerobic decarboxylation (mitochondrial matrix) = convert pyruvate (3 carbons) to an acetyl group (2 carbons).
      • 1 NADH made for every pyruvate.
      • Only occurs in the presence of oxygen.
      • Acetyl group attaches to Coenzyme A to make acetyl CoA.
    • Anaerobic fermentation (cytosol) = redox reaction: reduce pyruvate, oxidize NADH.
      • 1 NAD+ made for every pyruvate.
      • Alcohol fermentation = pyruvate reduced to ethanol.
      • Lactic acid fermentation = pyruvate reduced to lactate.
      • The purpose of anaerobic fermentation is to regenerate NAD+, which is needed for glycolysis.
  • Krebs cycle, substrates and products, general features of the pathway
    • Location: matrix of mitochondria.
    • Acetyl CoA feeds into the cycle.
    • 3 NADH made per acetyl CoA.
    • 1 FADH2 made per acetyl CoA.
    • 1 ATP (GTP) made per acetyl CoA.
    • Coenzyme A is regenerated (during the first step of the cycle).
    • Krebs cycle, TCA, Tricarboxylic acid cycle, citric acid cycle all mean the same thing.
    • Krebs cycle is Inhibited by ATP and NADH.
  • Electron transport chain and oxidative phosphorylation, substrates and products, general features of the pathway
    • Location: the cristae (inner membrane of mitochondria).
    • Input NADH
    • Proton gradient
    • The electron transport chain (ETC) is essentially a series of redox reactions, where NADH gets oxidized to NAD+ and O2 gets reduced to H2O.
    • The series of redox reactions consists of electrons passing from NADH to FMN, to Coenzyme Q, iron-sulfur complexes, and cytochromes (cytochrome b, c and aa3) before finally being used to reduce oxygen.
    • NADH is highest in energy, while O2 is lowest in energy. When electrons are passed from NADH down a series of proteins and finally to O2, energy is released.
    • FADH2 is lower in energy than NADH, that's why it releases less energy when it gets oxidized.
    • FADH2 skips FMN and passes its electrons to Coenzyme Q.
    • The energy released from these reactions generates a proton gradient, which drives ATP synthase to make ATP. This is called oxidative phosphorylation.
    • Proton gradient
      • The energy released from passing electrons down the ETC is used to pump protons into the intermembrane space of the mitochondria.
      • H+ concentration is very high in the intermembrane space (higher than those in the matrix). Thus, this establishes an electrochemical gradient called the proton gradient.
      • H+ wants to migrate down the proton gradient (from the intermembrane space back into the matrix), but it can only do this by going through the ATP synthase.
      • Like a water mill, ATP synthase harnesses the energy of the falling protons to convert ADP into ATP.
    • The ETC is inhibited by certain antibiotics, by cyanide, azide, and carbon monoxide.
  • Metabolism of fats and proteins
    • Fat metabolism
      • Location: beta-oxidation occurs in the matrix of the mitochondria. Ester hydrolysis occurs in the cytosol.
      • Fatty esters gets hydrolyzed into free fatty acids by lipases.
      • For example, triacylglycerol gets hydrolyzed into free fatty acids and glycerol.
      • With the help of ATP, the fatty acid is "activated" at the acid end by CoA (to be precise, it turns into a thioester).
      • A process called beta-oxidation breaks down the fatty-CoA, 2 carbons at a time, to make acetyl CoA.
      • β-oxidation produces acetyl CoA and also FADH2 and NADH.
      • The acetyl CoA feeds into the Krebs cycle, and the FADH2 and NADH feed into the ETC.
      • On a per gram basis, fats give more energy than any other food source.
    • Protein metabolism
      • Proteins are broken down into amino acids by peptidases.
      • The nitrogen in the amino acid is converted to urea (for desert animals, birds and reptiles, it is uric acid).
      • The carbon in the amino acid is converted to pyruvate or acetyl-CoA, (or other metabolical intermediates such as oxaloacetate), depending on what amino acid it is.
      • The carbon products from amino acid metabolism can either feed into the Krebs cycle, or be the starting material for gluconeogenesis.