CC2 LEC

• Factors that influence Enzymatic Reaction
• Enzymes

  • Proteins that catalyze chemical reactions
  • Biocatalysts that make chemical reactions faster
• Catalytic Mechanism of Enzymes

  1. A chemical reaction may occur spontaneously if the free energy or available kinetic energy is higher for the reactant than the products (lower energy)

  2. Activation Energy→ reactants have enough energy to break their bond and collide to form new bond (bond between enzyme and substrate)
• Enzymatic Reaction can be: First-Order Kinetic – the rate of reaction is directly proportional to the substrate concentration. Zero-order kinetic – the rate of reaction is dependent on the enzyme concentration. Saturation kinetic - even if we add more reactant to the reaction once the counterpart reactant has been saturated, it will no longer result in a fast reaction
• Co-factors are non-protein entities that must bind to an enzyme for a reaction to occur. Activators are made up of inorganic cofactors to alter the spatial configuration of the enzyme, e.g., metallic→ Ca, Fe, Mg, Mn, Zn, K, non-metallic→ Br, Cl. Coenzyme are organic cofactors to serve as a second substrate to enzyme reaction, e.g., Vitamins, AD (nicotinamide adenine dinucleotide); bridge between the enzyme and the substrate
• When the cell is injured, the enzyme will escape
• Temperature increase results in an increase in reaction because the molecules' movements are more active in colder temperatures
• Enzymes are highly specific

  • Absolute Specificity→ the enzyme combined with only one substrate and catalyzed single reaction
  • Group Specificity→ the enzyme combined with all substrates with a particular chemical group
  • Bond Specificity→ the enzyme combined with all substrates with a particular chemical bond such as hydrogen bond, etc.
  • Stereoisometric Specificity→ the enzyme combined with all substrate with the presence of optical isomer (mirror image)
  • Fast chemical reaction is the product of enzyme substrate complex
• Reactants

  1. Enzymes

  2. Active site - substrate binds the enzyme

  3. Allosteric site – free of water

  4. Substrate
• Factors that influence Enzymatic Reaction

  1. Substrate concentration/enzyme concentration

  2. Ph- 7-8pH

  3. Temperature

  4. Co-factors
• Enzymes are essential to physiologic functioning

  • Hydration of CO2 maintains the pH of the blood
  • Nerve conduction in the brain, e.g., CK-BB
  • Muscle contraction for muscular contraction, e.g., CK-MM
  • Nutrient degradation in the GIT, e.g., aminase, lipase
  • Energy use
  • Cardiac enzymes in the heart
  • Liver enzymes in the liver
  • Skeletal muscle enzymes
• Extreme pH can result in damage or breakdown of enzymes. Acid – ACP, Base – ALP
• Temperature coefficient – every 10°C increase results in a two-fold increase in the activity of the enzyme. 40-50°C→ start of denaturation of enzyme. 60-65°C→ inactivation of enzyme. 37°C (25 or 30°C) → optimum temperature of enzymatic reaction
• Storage

  1. Cold temperature can cause reversible inactivation of enzyme

  2. -20°C - long term preservation

  3. 2-8°C - general preservation

  4. Room Temperature - some enzymes are cold labile. Ex. LD (lactate dehydrogenase), LD4 and LD5

  5. Repeated thawing- putting in and out the enzyme forms the freezer can damage it
• Lactescence/Milky specimen


  Increasing concentration, low measurement. Chylomicrons
• Isoenzymes

  • Enzymes with the same catalytic function but different physical properties
  • CKBB (CK1) - brain
  • CKMM (CK3) - muscle
  • Differentiated by: Electrophoretic mobility, Solubility, Resistance to inactivation
• Types of Enzymes

  • Oxido-reductase - catalyze an oxidation-reduction reaction between two substrates
  • Transferase - catalyzed the transfer of a group other than hydrogen from one substrate to another
  • Hydrolase - catalyzed hydrolysis of various bonds
  • Lyase - catalyzed removal of the groups from substrate without hydrolysis; the product contains double bonds
  • Isomerase - catalyzed the interconversion of geometric, optical, or positional isomers
  • Ligases - catalyze the joining of the two substrate molecules, coupled with breaking of the pyrophosphate bond in adenosine triphosphate (ATP) or a similar compound
• Coenzyme


  • Organic cofactors; to serve as a second substrate to enzyme reaction
  • Ex. Vitamins, AD (nicotinamide adenine dinucleotide); bridge between the enzyme and the substrate
• Enzymes

  Proteins (Amino Acid)
• Inhibitors interfere with the chemical reaction

  1. Types of Inhibition: Competitive Inhibition - the target of the inhibitor is the active site

  2. Non-Competitive Inhibition - the target is the allosteric site

  3. Uncompetitive Inhibition - the target is the E.S complex
• Enzymology Part ii

  1. Creatine Kinase - Associated with ATP generation in the contractile system (muscle tissue)

  2. Function: in the muscle cells, it stores Creatine phosphate that is important in ATP production

  3. Tissue sources: Skeletal muscle, Heart muscle, Brain tissue

  4. Isoenzymes: Dimer with 2 subunits

  5. CK-1 - <1% aka CK-BB - brain type

  6. CK-2 - <6% CK-MB - hybrid type

  7. CK-3 - 94-100% CK-MM - muscle type (blood)

  8. Isoenzymes: by electrophoresis, we can differentiate CK

  9. CK-1= <1% - most anodal (anode)

  10. CK-2= <6%

  11. CK-3= 94-100% - most cathodal (cathode)
• Enzymatic Reactions

  1. 2 Methods in measuring the enzymatic reactions: Fixed-time - single measurement of the sample

  2. Continuous-Monitoring/Kinetic Assay - multiple measurement of enzyme activity
• Enzyme structure

  • Primary Structure - specific sequence of amino acid
  • Secondary Structure - polypeptide chain twisting
  • Tertiary Structure - folding of the polypeptide chain/secondary structure
  • Quaternary Structure - combination of tertiary structure
• Nomenclature-naming developed by the E.C. (Enzyme Commission)
• Alter the spatial configuration of enzyme

  1. Metallic: Ca, Fe, Mg, Mn, Zn, K

  2. Non-metallic: Br, Cl
• Hemolysis


  Destruction of RBC; K(Potassium) L(LDH) A(AST, ACP, aldolase) M(magnesium) P(phosphate) enzyme will increase during hemolysis
• Rise in CK-MB

  1. 8 hrs
• CK-1


  Less than 1%, most anodal (anode)
• CK-BB


  • Seldomly found in plasma, short half-life, high molecular size
• CK-3


  94-100%, most cathodal (cathode)
• Normalization of CK-MB

  48-72 hrs
• Macro CK


  Found midway between MM and MB
• Oliver-Rosalki Assay


  Measurement of Creatine Phosphate + ADP to Creatinine + ATP
• Methods used for the measurement of Isoenzymes of CK

  1. Electrophoresis

  2. Ion Exchange Chromatography

  3. Antibodies

  4. Immunoassay
• Lactate dehydrogenase catalyzes the interconversion of lactic acid and pyruvic acid
• For storage, CK can last up to 7 days at 4 degrees Celsius and 1 month at -20 degrees Celsius
• CK-MM

  • Major fraction in serum; skeletal muscle
  • Elevated in hypothyroidism, muscle activity, IM injection
• Peak in CK-MB

  12-24 hrs
• CK-2


  Less than 6%
• CK-MB

  • 20% of cardiac tissue contains CK-MB, specific to heart muscle, indicator of myocardial damage