Enzymes and Their Mechanisms

• Enzymes

  Proteins that speed up (catalyse) specific chemical reactions
• Enzymes

  • They are very important and without them, the body wouldn't be able to survive/function — reactions would occur too slowly
• Functions of Enzymes

  • Digestion: carbohydrates, fats, proteins
  • Blood clotting: fibrin clot catalysed by thrombin
  • Defence-immune system-activation of complement
  • Movement: muscle actomyosin is an ATPase
  • Nerve conduction: membrane pumps for Na⁺, K⁺, Ca²⁺
• Intracellular or Secreted Enzymes

  Enzymes are usually made inside the cell and they can be in an intracellular location or they can be secreted; this depends on the structure of the gene as to whether or not the protein has a tag that enables it to get out of the cell once it's been made in the ribosome
• Proteases


  Enzymes that have other proteins as their substrates and they're able to cleave peptide bonds
• Ribonuclease


  Enzymes that break down RNA and cleave nucleic acid
• Polymerase


  Adds basic building blocks together, e.g. DNA and RNA polymerase bring nucleotides or ribonucleotides together to make DNA or RNA
• Kinases


  Transfer a phosphate from ATP onto some other substrate and they change the structure of the substrate
• Inherited diseases involving an amino acid, a sugar and a complex lipid due to a lack of a particular enzyme

  • Phenylketonuria — can't convert phenylalanine to tyrosine due to a mutation in tyrosine hydroxylase
  • Glycogen storage disease — can't mobilise glucose, which is stored as a polymer called glycogen in the muscle
  • Tary-Sachs disease — defect in processing a membrane ganglioside, which is a complicated sort of lipid, which is important in the brain for its membrane function

  
• Enzymes

  • Increase reaction rate by up to 10 billion fold
  • Show specificity — they don't speed up the rates of all reactions, they just have a very particular reaction that they work on
  • Unchanged at end of reaction — they can participate in the reaction chemically by acting as a template to bind the components of the reaction and allow them to react, but they can also participate in the reaction directly
  • Do not alter reaction equilibrium between the substrate and the product (thermodynamics, not kinetics) — the reaction equilibrium between the substate and the product is fixed by the structures of the reactants and products
  • Facilitate reaction by decreasing the free energy of activation of the reaction

  
• Active Site

  A 3D cavity or cleft that binds substrate(s) with specificity through electrostatic, hydrophobic, hydrogen bonding and van der Waals interactions
• Formation of an enzyme-substrate ES complex at the active site

  The first step in enzyme catalysis. Not available in the uncatalyzed reaction
• Lock and Key vs Induced Fit Mechanisms

  Lock and Key: The enzyme's active site has a particular shape and it binds a substrate, which is complimentary to that, i.e. has the same shape

  Induced Fit: The active site doesn't look completely like the substrate, but as the substrate begins to bind, the active site becomes complementary in shape to the substrate

  
• Factors Responsible for Enzyme Catalysis

  • Bring molecules together in the active site

  Constrain substrate movement

  Strain particular bonds in the substrate that's necessary for the reaction to work, making breakage easier

  Stabilise positive and negative charges in the transition state

  Exclude water from the active site, making reaction go faster

  Provide a reaction pathway of lower energy e.g. involving covalent enzyme-substrate intermediates

  Use cofactors: bring new chemistry to the active site with NADH, FADH₂, metal ions such as Mg²⁺

  
• Enzyme Kinetics — Vmax and Km

  Vmax is the maximum rate of the reaction, Km is the substrate concentration at which the reaction rate is half of Vmax. Vmax/[enz] gives the turnover number (kcat), which is the max no. of substrate molecules handled per active site per second
• Competitive Inhibition


  Competitive inhibitors look like the substrate for a particular enzyme. They block the binding of the substrate by interacting themselves with the active site. This means if the inhibitor is bound, the substrate can't bind. In the presence of a competitive inhibitor, Km is increased but Vmax is unaltered

  
• Non-competitive Inhibition

  Inhibitor I binds at a different site and does not compete with substate S for binding at the active site. Therefore, in the presence of a non-competitive inhibitor, the Km is unaltered but the Vmax is reduced

  
• Ways enzyme activity is regulated in cells

  • Control of gene expression — enzyme amount: making more of a protein that's important for the cell under those conditions

  Compartmentation — sequences in enzyme polypeptide chain target enzyme to ER, mitochondrion, nucleus etc, making sure it goes to the place its supposed to be doing its job

  Allosteric regulation — a regulatory molecule (acting at a pocket distinct from the active site) changes the enzyme conformation to influence the active site and decrease (or in some cases, increase) enzyme activity

  Covalent modification of enzyme, which changes enzyme shape and activity, e.g. phosphorylation
• Feedback inhibition

  The product looks nothing like the substrate when it's formed, meaning it's unable to bind directly to the active site. So it comes back and inhibits the enzyme by binding to a different site, changing the shape of the enzyme and results in a sort of negative feedback of the flux through the pathway

  
• Control of glycolysis achieved by allosteric regulation — including the early enzyme phosphofructokinase which is regulated by citrate, ADP and ATP