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
- 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.
- 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.