Enzymes

• Enzymes


  • Protein catalysts
  • Some small RNAs (ribosimes) have a similar function
  • Catalyze specific biochemical reactions
  • Almost every reaction in a living organism requires an enzyme
  • They organize, coordinate, regulate the cell metabolism
• Why study enzymes


  • Genetic disorders: amount of enzyme; changes in the enzyme kinetics; abnormal enzyme
  • Drugs: enzymes, enzyme inhibitors
  • SNPs in the genes coding for certain enzymes – drug metabolism variations
  • Enzyme-based diagnostic tests in clinical practice
  • Diseases are diagnosed by the enzyme activities
  • Nutrition: enzyme deficiencies o nutritional deficiencies; personalized diets
• General properties of enzymes

  • Increase the velocity of a spontaneous chemical reaction without being changed in the overall process; they change the reaction equally in both directions, and they do not change the equilibrium of the reaction
  • High catalytic activity
  • They function in solutions, at physiological pH, T and in negligible amounts
  • Highly specialized catalysts
• Enzymes are highly specific


  • Reaction specificity: One and the same substrate o different enzymes o different products
  • Substrate specificity
• General characteristics of enzymes

  • Active center: unique, three-dimensional
  • Regulated
  • Specific localization
• Cell localization of enzymes

  1. Cytosol: glycolysis, PPP, FA synthesis

  2. Mitochondria: Krebs cycle, FA oxidation, decarboxylation of pyruvate

  3. Lysosomes: degradation of biomolecules

  4. Nucleus: DNA and RNA synthesis

  
• Holoenzyme


  Apoenzyme + non protein compound (cofactor)

  
• Cofactors


  • Coenzymes
  • Prosthetic groups
• Nomenclature


  • Trivial names
  • Suffix "-ase"
  • Nomenclature system of the International Union of Biochemistry and Molecular Biology (IUВMB)
• Chemical elements that function as prosthetic groups in enzymes

  • Cobalt
  • Copper
  • Iron
  • Molybdenum
  • Selenium
  • Zinc

  
• Cofactors involved in group transfer

  • Nucleosidephosphates (Pi, PPi)
  • Coenzyme А (acyl groups)
  • Thiamine pyrophosphate (hydroxyl)
  • Pyridoxal phosphate (amino groups, AA residues)
  • Biocytin (СО2)
  • Tatrahydrofolate (С1)
  • Methylcobalamine, 5' deoxyadenosylcobalamin (СН3-)

  
• Cofactors involved in proton and electron transfers

  • NAD(P)+
  • Flavine coenzymes: FMN, FAD
  • Lipoamid
  • Glutathione (GSH)
  • Ubiquinone
  • Ascorbic acid
  • Heme

  
• Vitamins


  • Chemically unrelated organic compounds
  • They cannot be synthesized by humans, must be supplied by the diet
  • Needed in minor amounts
  • Required to perform specific cellular functions
  • They are precursors of coenzymes
  • Classified based on their solubility and functions

  
• Vitamin C


  • Source: green vegetables, potatoes, rose hips, black current, tomatoes, peppers, citrus fruits
  • Deficiency: fragile blood vessels, scurvy in more severe deficiencies (deficiency in the hydroxylation of collagen, resulting in defective connective tissue)
  • Role – reducing agent: Coenzyme in hydroxylation reactions, facilitates the absorption of dietary iron, important antioxidant, maintains metal ions in reduced state, role in the catabolism of tyrosine
• Vitamin C - clinical aspects

  • May have a role in the prevention of atherosclerosis and cancer
  • Role in immunity response
• Vitamins complex B


  • Thiamine (В1)
  • Riboflavin (В2)
  • Niacin (В3)
  • Pantothenic acid (В5)
  • Pyridoxine (В6)
  • Biotin (B7)
  • Folic acid (B9)
  • Cobalamin (В12)

  
• Thiamin (В1)


  • Biologically active form: thiamin pyrophosphate
  • Role: Coenzyme in the formation or degradation of α-ketols by transketolase (non-oxidative reactions of PPP) and in the oxidative decarboxylation of D-keto acids (pyruvate DH complex; D-KG DH complex)
  • Source: beans and wheat, meat, liver, yolk
  • Deficiency: Beriberi, Wernicke-Korsakoff syndrome

  
• Riboflavin (В2)


  • Biologically active forms: FMN, FAD
  • Role: Coenzyme in oxidation and reduction reactions, prosthetetic group of flavoproteins
  • Source: grains, milk, meat, eggs
  • Deficiency: Riboflavin deficiency (cheilosis, glossitis, and seborrheic dermatitis) is not associated with a major human disease, although it frequently accompanies other vitamin deficiencies
• Niacin (nicotinic acid) – vitamin B3

  • Biologically active forms: NAD+, NADP+
  • Role: Oxidation-reduction reactions, Biosynthetic reactions (NADP+)
  • Source: grains and cereal, milk, lean meats, especially liver, peanuts, limited synthesis from tryptophan
  • Clinical aspects: Pellagra, Therapeutic agent in type IIb hyperlipoproteinemia, High doses – transient hepatotoxicity

  
• Pantothenic acid (B5)


  • Role: Central role in acyl group metabolism when acting as the pantetheine functional moiety of coenzyme A or acyl carrier protein (ACP)
  • Source: widely distributed in all foodstuffs
• Pantothenic acid (B5)


  Vitamin B5

  
• Pyridoxine (B6)


  Vitamin B6

  
• Folic acid (B9)


  Vitamin B9
• Cobalamin (B12)


  Vitamin B12
• Biotin (B7)


  Vitamin B7
• Water-soluble vitamins


  • Their biologically active forms are coenzymes
  • Antioxidants (reducing properties of vitamin C)
  • Toxicity due to greater intake is rare (B6)
  • Easy to be excreted due to their solubility in water
  • With some exceptions (B12) there are no depots of water-soluble vitamins in the body

  
• Enzyme kinetics


  Studies reaction velocity and the factors affecting it

  
• Reaction velocity (V)


  The number of substrate molecules converted to product per unit time

  
• Enzyme kinetics


  1. Velocity: Information about the concentration/activity of the enzyme

  2. Reaction velocity changes with the time

  3. Kinetics curves: Changes with the time

  
• Factors affecting reaction velocity

  • Substrate concentration
  • Inhibitors and activators
  • pH
  • Temperature

  
• Reaction velocity increases with substrate concentration


  Until a maximal velocity is reached

  
• Conditions for measuring initial reaction velocity

  • [S] >>> [E]
  • V is measured as soon as enzyme and substrate are mixed
  • The rate limiting step is ES -> E + P

  
• Michaelis-Menten equation


  Relates initial velocity to [S] and Vmax through the constant Km
• Reaction order


  • At [S] >>> Km, the reaction is zero order: V depends only on [E], equals to Vmax and is constant
  • At [S] << Km, the reaction is first order with respect to S: V is proportional to [S]
• Km


  Reflects the affinity of the enzyme for a substrate

  
• Vmax


  Depends on the enzyme concentration

  
• Enzyme activity units


  • Katal [kat] = 1mol S/1 sec, 25o C
  • 1U = 1 µmol S/1 min
  • 1U = 16.67 kat
  • Relative enzyme activity: U/1 mg protein

  
• Effect of pH on enzyme velocity

  Ionization of the active site, denaturation

  
• Effect of temperature on enzyme velocity

  Toptimum depends on: pH, Oxidation-reduction potential, Additives, Broader in a dry state

  
• Measuring hexokinase activity


  1. Determine V to determine enzyme activity and thus enzyme amount

  2. Need to know: Summary equation, Suitable analytical approach, Km, pH optimum, T optimum