Clinical Chemistry

• Enzymes


  • Specific biologic proteins that catalyze biochemical reactions without altering the equilibrium point of the reaction
  • Neither consumed nor permanently altered as a consequence of their participation in a reaction
• Active site

  • Water free cavity where the substrates interact with the particular charged amino residues
  • A 3D protein structure
• Essential functions of enzymes

  • Hydration of carbon dioxide
  • Nerve conduction
  • Muscle contraction
  • Nutrient degradation
  • Energy storage and use
• Terms associated with Enzymes


  • Active site
  • Allosteric Site
  • Substrate
  • Isoenzyme
  • Apoenzyme
  • Holoenzyme
  • Cofactors
  • Proenzyme or Zymogens
  • Allosteric enzymes
• Allosteric Site


  Another cavity than the active site that binds regulatory molecules
• Substrate

  Substance acted upon by enzymes specific for each of their particular enzyme
• 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
• Cofactors


  Non-protein substances added in the enzyme-substrate complex to manifest the enzyme activity
• Types of Cofactors

  • Coenzyme or Prosthetic group
  • Activator
• Coenzyme or Prosthetic group


  • Second substrates for enzymatic reactions
  • Increasing coenzyme concentration will increase the velocity of an enzymatic reaction in a manner synonymous with increasing substrate concentration
  • An organic cofactor
• Coenzyme or Prosthetic group

  • Nicotinamide adenine dinucleotide(NAD)
  • Nicotinamide adenine dinucleotide phosphate (NADP) reduced form (Primary substrate is oxidized)
• Proenzyme or Zymogens


  • An inactive enzyme precursor
  • Examples: Coagulation factors, Digestive enzymes
• Activator

  • An inorganic cofactor
  • Metallic ions: (Ca2+, Fe2+, Mg2+, Mn2+, Zn2+, and K+)
  • Nonmetallic ions (Br− and Cl−)
• Allosteric enzymes


  • Regulator of cellular processes, but not all enzymes are allosteric
  • Some can be allosteric provided that they are composed of quaternary structures with two or more protein chains containing the active sites and regulatory sites (binding sites)
  • The substances that bind on the regulatory sites are called Regulator
• Two kinds of allosteric enzymes

  • Homoallostery
  • Heteroallostery
• Homoallostery


  Cooperative substrate binding and activation wherein substrate is a homotropic effector. Therefore, the binding of substrate to one active site alters the substrate binding affinity and/or catalytic activity at other active sites on the multimeric enzyme
• Heteroallostery

  Involves the regulation by heterotropic effector molecules, which can be positive (activation) or negative (inhibition). These effectors usually bind at a site other than the active site and can activate or inhibit the activity of an enzyme
• Enzyme Classification and Nomenclature

  The system for the classification of enzymes that also serves as a basis for assigning code numbers to them
• Hydrolases


  • Catalyze hydrolysis of various bonds
• Transferases


  • Catalyze the transfer of a group other than hydrogen from one substrate to another
• Ligases


  • Catalyze the joining of two substrate molecules, coupled with breaking of the pyrophosphate bond in adenosine triphosphate (ATP) or a similar compound
• Enzyme Classification and Nomenclature

  1. The system for classification of enzymes that also serves as a basis for assigning code numbers to them

  2. These code numbers, prefixed by EC, contain four elements separated by points, with specific meanings

  3. The first number shows to which of the six main divisions (classes) the enzyme belongs

  4. The second number indicates the subclass

  5. The third number indicates the sub-subclass

  6. The fourth number is the serial number of the enzyme in its sub-subclass
• Isomerases


  • Catalyze the interconversion of geometric, optical, or positional isomers
• Oxidoreductases


  • Catalyze an oxidation-reduction reaction between two substrates
• Lyases


  • Catalyze removal of groups from substrates without hydrolysis; the product contains double bonds
• Factors That Influence Enzymatic Reactions
• pH


  1. Changes in pH may denature an enzyme or influence its ionic state, resulting in structural changes or a change in the charge on an amino acid residue in the active site

  2. pH range: 7.0 – 8.0
• Temperature

  1. Optimal temperature: 37°C

  2. Denaturation: 40-50°C

  3. Incubation temperatures: within ± 0.1°C

  4. Routine enzyme measurement temperatures: 25°C, 30°C, 37°C

  5. Freezing and Thawing of specimen for measuring of enzyme activity should be avoided
• Substrate Concentration

  1. Rate at which an enzymatic reaction proceeds and whether the forward or reverse reaction occurs depend on several reaction conditions

  2. Michaelis and Menten hypothesis in 1913: The role of substrate concentration in the formation of the enzyme–substrate (ES) complex

  3. First-order kinetics: The reaction rate is directly proportional to substrate concentration

  4. Zero-order Kinetics: The reaction rate depends only on enzyme concentration
• Enzyme Concentration

  1. The enzyme concentration affects the rate of the catalyzed reaction

  2. The higher the enzyme level, the faster the reaction will proceed because more enzyme is present to bind with the substrate
• Cofactors

  1. Nonprotein entities that must bind to particular enzymes before a reaction occurs

  2. Activators: Metallic ions and nonmetallic ions

  3. Coenzymes: Nucleotide phosphate and vitamins
• Inhibitors

  1. Competitive inhibitors physically bind to the active site of an enzyme and compete with the substrate for the active site

  2. If the substrate concentration is significantly higher than the concentration of the inhibitor, the inhibition is reversible because the substrate is more likely to bind
• Measurement of Enzyme Activity

  1. Quantitation method based on enzymatic activity is the measurement of catalytic activity

  2. Methods include increase in product concentration, decrease in substrate concentration, increase or decrease in coenzyme concentration (NADH), increase in altered enzyme concentration

  3. Dependent on enzyme concentration

  4. Performed in zero-order kinetics (linear phase)
• Competitive inhibitors

  1. Physically bind to the active site of an enzyme and compete with the substrate for the active site

  2. If the substrate concentration is significantly higher than the concentration of the inhibitor, the inhibition is reversible because the substrate is more likely than the inhibitor to bind the active site and the enzyme has not been destroyed
• Noncompetitive inhibitor

  1. Binds an enzyme at a place other than the active site

  2. Some naturally present metabolic substances combine reversibly with certain enzymes

  3. If the inhibitor destroys part of the enzyme involved in catalytic activity, the inhibition is irreversible

  4. The inhibitor binds the enzyme independently from the substrate, increasing substrate concentration does not reverse the inhibition
• Methods of measuring enzymatic reaction

  1. Fixed time (Two point) Assay

  2. Continuous-monitoring or kinetic assays
• Uncompetitive inhibition

  1. Bind to the Enzyme substrate complex

  2. Increasing substrate concentration results in more Enzyme Substrate complexes to which the inhibitor binds and, thereby, increases the inhibition
• Units used for enzymatic activity

  1. International Unit: 1 μmol of substrate/minute

  2. Katal Unit: 1 mole of substrate/second

  3. Enzyme concentration is usually expressed in units per liter (IU/L)

  4. Enzyme activity measurements may not be accurate if enzyme inhibitors are present, essential cofactors are not included in the assay, and improper storage of specimen

  5. Enzymes are never reported in concentration, only based on their activity
• Major Clinical Significance of Enzymes