L7

• Enzymatic reactions


  • Chymotrypsin
  • Hexokinase
  • Enolase
  • Lysozyme

  
• Chymotrypsin


  A serine protease
• Understanding the mechanism of enzymes: Catalytic mechanism

  1. Identify all substrates, cofactors, products, and regulators

  2. Know the sequence in which enzyme-bound reaction intermediates form

  3. Know the structure of each intermediate and each transition state

  4. Know the rates of interconversion between intermediates

  5. Know the structural relationship of the enzyme to each intermediate

  6. Know the energy contributed by all reacting and interacting groups to the intermediate complexes and transition states

  
• Chymotrypsin


  • Secreted by the pancreas
  • Breaks down proteins in the small intestine
  • Activated by trypsin
  • Catalyzes the hydrolytic cleavage of peptide bonds
  • Specific for peptide bonds adjacent to aromatic amino acid residues: Phe, Tyr, Trp
  • Enhances peptide bond hydrolysis by 10^9
  • Catalysis is via a transient covalent acyl enzyme intermediate

  
• Chymotrypsin is made up of 3 polypeptides (A, B, C) joined by disulfide bonds

  
• Zymogen


  A proenzyme, an inactive precursor of an enzyme

  
• Chymotrypsin-activation of zymogens


  1. Biochemical change occurs in Golgi bodies, where a specific part of the precursor enzyme is cleaved in order to activate it

  2. Activated by trypsin which cleaves it

  
• Blood coagulation is a system in which a series of zymogens (enzyme precursors) of serine proteases are sequentially activated

  
• Hemophilia A is a disorder related to blood coagulation
• Chymotrypsin reaction mechanism


  1. Involves a covalent acyl enzyme intermediate

  2. Evidence from pre-steady state kinetics

  3. Initial rapid burst of product release

  4. Subsequent slower rate due to enzyme turnover limited by deacylation

  
• Catalytic triad


  Key active-site residues in chymotrypsin including Ser195, His57, and Asp102

  
• Mechanism of action of chymotrypsin

  1. Active-site serine residue attacks the carbonyl group of the peptide bond

  2. Carboxy-terminal peptide is released, amino-terminal peptide remains as enzyme-bound acyl intermediate

  3. Ester bond is hydrolyzed to release amino-terminal peptide and regenerate active enzyme

  
• Types of Proteases


  • Serine proteases (Chymotrypsin, Trypsin, Elastase)
  • Cysteine proteases (Papain, Caspases)
  • Threonine proteases (Proteasome)
  • Aspartyl peptidases (Renin, Pepsin, HIV proteases)
  • Metalloproteases (Carboxypeptidase)

  
• Serine proteases


  Enzymes that cleave peptide bonds, with serine as the nucleophilic amino acid at the active site
• Acetylcholinesterase is an important serine protease
• Insecticides (organic fluorophosphates) can form covalent complexes with acetylcholinesterase, inhibiting the enzyme

  
• Atropine is a competitive inhibitor of acetylcholinesterase
• When acetylcholinesterase is depleted, acetylcholine neuroactivation is prolonged and sustained, leading to inability to neuromuscular stimulation and breathlessness
• HIV protease


  An aspartyl protease essential for the HIV life cycle

  
• Mechanism of action of HIV protease

  1. 2 active-site aspartate residues facilitate the attack of water on the peptide bond

  2. Cleaves peptide bonds between Phe and Pro residues

  
• HIV protease inhibitors


  Transition state analogs that bind tightly to the HIV protease active site

  
• PAXLOVID is an investigational SARS-CoV-2 protease inhibitor antiviral therapy
• PAXLOVID is designed to block the activity of the SARS-CoV-2-3CL protease

  
• Lysozyme is an antibacterial agent found in tears and egg white
• Lysozyme


  • Can breakdown the peptidoglycan in the bacterial cell wall
  • Also called muramidase
  • First enzyme to have its 3D structure determined

  
• Lysozyme cleaves the (β1 → 4) glycosidic C—O bond between the 2 types of polysaccharides (NAG and NAM) in peptidoglycan

  
• ARS-CoV-2-3CL protease


  An enzyme that the coronavirus needs to replicate

  
• Co-administration with a low dose of ritonavir

  Helps slow the breakdown of PF-07321332
• RAAS

  Renin Angiotensin-Aldosterone system, regulator of blood pressure and electrolyte
• ACE inhibitor


  Leads to vessel dilation, Lowering of blood pressure

  
• Lysozyme


  An antibacterial agent found in tears and egg white
• Lysozyme


  • It can breakdown the peptidoglycan in the bacteria cell wall
  • It is also called muramidase

  
• Hen egg white lysozyme

  A monomer with 129 amino acid residues, 1st enzyme to have its 3D structure determined (David Phillips et al 1965)
• Lysozyme

  • Has 4 stabilizing disulfide bonds and a cleft containing the active site

  
• Peptidoglycan


  The substrate for lysozyme, a carbohydrate (with amino acids) found in many bacterial cell walls

  
• Lysozyme action


  Cleaves the (β1 → 4) glycosidic C—O bond between the 2 types of sugar residues in the molecule, N-acetylmuramic acid (Mur2Ac) and N-acetylglucosamine (GlcNAc)

  
• The (β1 → 4) glycosidic C—O bond is cleaved by lysozyme

  
• Glycan


  Polymers of sugar molecules (polysaccharides or carbohydrates) joined together by glycosidic bonds
• Lysozyme active site


  • 6 residues of the alternating NAM and NAC bind in the active site, in binding sites designated A through F
  • Only 1 of the bound glycosidic bonds is cleaved, that which is between a Mur2Ac residue in site D and a GlcNAc residue in site E
  • The key catalytic amino acid residues in the active site are Glu35 and Asp52

  
• The glycosidic C—O bond between sugar residues bound to sites D and E is cleaved by lysozyme