Enzymology

• Enzyme functions:

  • Hastens chemical reactions in organic matter
  • Catalyzes single or limited number of chemical reactions
  • Large molecules confined in cells
  • Increase membrane permeability allows them to enter the blood
  • Measured in terms of their activity and not of their absolute values
  • Appear in serum after cellular injury, degradation of cells or from storage areas
  • Used clinically as evidence of organ damage
  • Specific for substrate that it converts to a defined product
• Enzymes and their functions:

  1. Hydration of CO2 (respiration) – Carbonic Anhydrase

  2. Nerve induction – Choline acetyltransferase (Cholinergic enzymes)

  3. Muscle contraction – AChE

  4. Nutrient degradation (digestion): Pepsin, Amylase, Lipase

  5. Growth and reproduction: Helicase (Cell division)

  6. Energy storage and use: LDH, PPK1 & PPK2 (Conversion and conservation of ATP)
• Components of an enzyme:

  • Active Site: A cavity of an enzyme where substrates bind and undergo a chemical reaction
  • Allosteric Site: A cavity other than the active site that binds regulatory molecules
• Terms associated with an enzyme:

  • Substrates: Substances acted upon by enzymes, specific for their particular enzyme
  • Cofactors: Substance added in the enzyme-substrate complex to manifest the enzyme activity
  • Isoenzyme: Similar enzymatic activity but differ in physical, biochemical, and immunologic characteristics
  • Apoenzyme: The protein portion of the enzyme, subject to denaturation, in which enzyme loses its activity
  • Holoenzymes: An active substance formed by combination of a co-enzyme and an apoenzyme
  • Proenzyme or Zymogens: An inactive enzyme precursor
• Enzyme classification and nomenclature:

  1. Oxidoreductases: Catalyze redox reactions between 2 substrates

  2. Transferase: Catalyze the transfer of a group between 2 substrates

  3. Hydrolases: Catalyze hydrolysis of various bonds

  4. Lyases: Removal of groups from substrates without hydrolysis; product remains double bonds

  5. Isomerases: Interconversion of geometric, optical, or positional isomers

  6. Ligases: Joins 2 substrate molecules, coupled with breaking of pyrophosphate bond in ATP
• Enzyme kinetics:

  • Enzymes catalyze physiologic reactions by lowering the activation energy level that the reactants must reach
  • Rate of reaction is proportional to the concentration of molecules that have attained energy equal to the free energy of activation
  • Michaelis-Menten constant: Describes the relationship between enzyme, substrate, and product
• Total enzyme concentration in serum and other body fluids can be determined using Product O
• Enzyme (E) catalyzes a reaction by combining with its substrate (S) to create an enzyme-substrate complex (ES)
• Types of specificity:

  • Absolute specificity: enzymes combine with only one substrate and catalyze only one corresponding reaction
  • Group specificity: enzymes combine with all substrates containing a particular chemical group
  • Bond specificity: enzymes are specific to chemical bonds
  • Stereoisomeric specificity: enzymes predominantly combine with only one optical isomer of a certain compound
• Emil Fisher's Lock and Key Theory:

  • Based on the rigid enzyme molecule into which the substrate fits
  • Absolute specificity: enzymes combine with only one substrate and catalyze only one corresponding reaction
• Factors that influence enzymatic reactions:

  A. Substrate concentration:

  • First order kinetics (Michaelis-Menten): reaction rate is proportional to substrate concentration
  • Saturation kinetics: rate of reaction reaches a maximal value at a particular concentration of substrate

  B. Enzyme concentration:

  • Zero order kinetics: only a fixed number of substrate is converted to product per second
  • An increase in enzyme concentration increases the rate of reaction
• pH:

  • Rate of any chemical reaction is usually increased 2-3 times for every 10 degrees Celsius rise in temperature
  • Optimal pH is 7.0-8.0, controlled by buffers
• Temperature:

  • Should be within ±0.1°C (e.g., 25°C, 30°C, 37°C: optimal temp.)
• Cofactors:

  • Essential ions include K, Ca, Mg, Na, Mn, Fe, Co, Se, Zn, and Cu
  • Coenzymes include derivatives of B complex vitamins and fat-soluble vitamins
  • Coenzymes can be ATP, S-adenosyl-methionine, ascorbic acid, NAD, NADP, tetrahydrofolate, coenzyme A, retinal, and vitamin K
  • Prosthetic groups include FAD, FMN, pyridoxyl phosphate, biotin, adenosylcobalamin, and lipoamide
• Inhibitors:

  • Reversible reactions:
  • Competitive Inhibitor: substances that compete with the substrate for enzyme binding
  • Non-Competitive Inhibitor: substances that do not resemble the substrate and bind to the enzyme in areas other than the active site
  • Irreversible reactions:
  • Uncompetitive inhibition: inhibits enzyme by binding to the enzyme-substrate complex
  • Irreversible Inhibitor: acts as a substrate analog and forms covalent bonds with the enzyme at the active site
• Salts:

  • The ionic strength of solutions affects enzyme activity
• Proteins:

  • Enzyme activity over the linear limits of the assay may require dilution of plasma with enzyme diluents containing plasma proteins
• Measurement of enzyme activity:

  • Methods include measuring catalytic activity through changes in product concentration, substrate concentration, coenzyme concentration, enzyme concentration, and performed in zero-order kinetics
• Units for expressing enzyme activity:

  • International Unit (I.U. or U): catalyzes the conversion of 1 micromole of substrate per minute
  • Katal Unit (K.U.): catalyzes the conversion of 1 mole of substrate per second
• Pitfalls in enzyme assays:

  • Hemolysis can lead to falsely evaluated values due to the release of enzymes from red blood cells
  • Serum is preferred over plasma due to the adverse effects of anticoagulants on enzyme activity
  • Lactescent or milky serum can cause variable absorption by the spectrophotometer
• Storage of samples:

  • Most enzymes are stable at 6 degrees Celsius for at least 24 hours
  • Few enzymes are inactivated at refrigerator temperature
• Importance of studying enzymes:

  • Enzymes are essential for life and mutations in enzyme genes can cause diseases
  • Enzymes are targets for therapeutic drugs and have various uses and applications
• Mutation on genes coding for enzymes can cause deficiency diseases:

  • Phenylketonuria: Phenylalanine hydroxylase
  • Albinism: Tyrosinase
  • Maple syrup urine disease: alpha-keto acid dehydrogenase
  • Gaucher's disease: Glucocerebrosidase
  • Niemann-Pick's disease: Sphingomyelinase
  • Tay Sach's disease: Hexosaminidase A
• Enzymes in clinical diagnosis:

  • Elevated serum enzyme levels can help diagnose specific diseases