Enzymes

Cards (66)

  • 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