enzyme

• Enzymes


  Protein catalysts that increase the rate of reactions without themselves being changes in the overall process
• Enzymes


  • They virtually mediate all reactions in the body
  • They selectively channel reactants (substrates) into useful pathways
  • They direct all metabolic events
• Nomenclature


  Each enzyme is assigned two names: a short, recommended name for everyday use and a more complex systematic name used for identification
• Recommended enzyme names


  • Names with the suffix "-ase" attached to the substrate of the reaction (e.g. glucosidase, urease, sucrase)
  • Names that describe the action (e.g. lactate dehydrogenase, adenylate cyclase)
• Some enzymes retain their original trivial names, which give no hint of the associated enzymic reaction (e.g. Trypsin, pepsin)
• Systematic enzyme names


  • Developed by the International Union of Biochemistry and Molecular Biology (IUBMB)
  • Enzymes are divided into six major classes, each with numerous subgroups
  • The suffix "-ase" is attached to a complete description of the chemical reaction catalyzed (e.g. D-glyceraldehyde 3-phosphate: NAD oxidoreductase)
• Six major classes of enzymes

  • Oxidoreductases
  • Transferases
  • Hydrolases
  • Lysases
  • Isomerases
  • Ligases
• Active sites


  • Enzyme molecules contain a special pocket or cleft called the active site, which contains amino acid side chains that create a three-dimensional surface complementary to the substrate
  • The molecule that is bound in the active site and acted upon by the enzyme is called the substrate
  • The substrate binds the substrate forming an enzyme-substrate (ES) complex, which is converted to an enzyme-product (EP) complex that then dissociates to release the enzyme and product

  
• Catalytic efficiency


  • Most enzyme-catalyzed reactions are highly efficient, 10 times faster than uncatalyzed reactions
  • Their catalytic activity depends on the integrity of their native protein conformation
  • The catalytic activity of each enzyme is intimately linked to its primary, secondary, tertiary, and quaternary protein structure
  • Turnover number is the number of molecules of substrate converted to product per enzyme molecule per second
• Specificity


  • Enzymes are highly specific, interacting with one or a few specific substrates and catalyzing only one type of chemical reaction
• Cofactors


  • Other enzymes require an additional chemical component called a cofactor (either one or more inorganic ions or a complex organic or metalloorganic molecule called a coenzyme)
  • Common cofactors include metal ions (like Zn2+, Fe2+) and organic molecules known as coenzymes that are often derivatives of vitamins (NAD+, FAD, coenzyme A)
  • Holoenzyme is the enzyme with its cofactor, while apoenzyme is the protein portion of the holoenzyme that does not show biological activity in the absence of the appropriate cofactor
  • A prosthetic group is a tightly bound coenzyme that does not dissociate from the enzyme (e.g. Biotin of carboxylases)
• Regulation


  Enzymes can be activated or inhibited so that the rate of product formation responds to the needs of the cell
• Location within the cell

  Many enzymes are located in specific organelles within the cell, which serves to isolate the reaction of the substrate or product from other competing reactions, provide a favorable environment for the reaction, and organize the enzymes into purposeful pathways (e.g. Mitochondria - TCA cycle, Fatty acid oxidation; Cytosol - Glycolysis, HMP pathway; Nucleus - DNA and RNA synthesis; Lysosome - Degradation of complex macromolecules)

  
• Enzymatic action - Energy changes

  • Enzymes provide an alternate, energetically favorable reaction pathway different from the uncatalyzed reaction
  • The free energy of activation is the energy difference between the reactants and a high-energy intermediate that occurs during the formation of the product
  • The lower the free energy of activation, the more molecules have sufficient energy to pass over the transition state, the faster the rate of the reaction
• Enzymatic action - Active site chemistry


  • The active site acts as a flexible molecular template that binds the substrate in a geometry structurally resembling the activated transition state of the molecule, stabilizing the transition state and accelerating the reaction
  • The active site can also provide catalytic groups that enhance the probability of the transition state forming, such as through general acid-base catalysis or transient covalent enzyme-substrate complex formation

  
• The enzyme-catalyzed conversion of substrate to product can be visualized as being similar to removing a sweater from an uncooperative infant, where the enzyme guides the substrate through the transition state to facilitate the reaction

  
• Factors affecting reaction velocity

  • Substrate concentration - Reaction rate increases with substrate concentration until a maximal velocity (Vmax) is reached
  • Temperature - Reaction velocity increases with temperature until a peak is reached, then decreases due to enzyme denaturation
  • pH - The concentration of H+ affects the ionization of the active site and can also lead to enzyme denaturation at extremes
• Michaelis-Menten equation


  Describes how reaction velocity varies with substrate concentration: Vo = Vmax(S) / (Km + S), where Vo is the initial reaction rate, Vmax is the maximal velocity, Km is the Michaelis constant, and (S) is the substrate concentration

  
• The Michaelis-Menten model assumes the concentration of substrate (S) is much greater than the concentration of enzyme (E)
• pH at which maximal enzyme activity is achieved


  Reflects the H+ at which an enzyme functions in the body
• Michaelis-Menten Equation


  Enzyme + Substrate ⇌ Enzyme-Substrate Complex → Enzyme + Product

  
• Michaelis-Menten model


  Accounts for most features of enzyme-catalyzed reactions

  
• In the Michaelis-Menten model, the enzyme reversibly combines with its substrate to form an ES complex that subsequently breaks down to product, regenerating the free enzyme
• Michaelis-Menten equation


  Describes how reaction velocity varies with substrate concentration

  
• Vo = Vmax (S) / (Km + (S))
• Assumptions of Michaelis-Menten kinetics:
• Michaelis constant (Km)


  • Characteristic of an enzyme and a particular substrate, reflects the affinity of the enzyme for that substance, numerically equal to the substrate concentration at which the reaction velocity is equal to ½ Vmax, does not vary with the concentration of enzyme

  
• Small Km


  Reflects a high affinity of the enzyme for substrate because a low concentration of substrate is needed to half-saturate the enzyme, velocity that is ½ Vmax
• Large Km


  Reflects a low affinity of enzyme for substrate because a high concentration of a substrate is needed to half-saturate the enzyme
• The rate of the reaction is directly proportional to the enzyme concentration at all substrate concentrations
• Order of reaction


  When (S) is much less than Km, the velocity of the reaction is roughly proportional to the substrate concentration (first order with respect to substrate)

  When (S) is much greater than Km, the velocity is constant and equal to Vmax (zero order with respect to substrate concentration)

  
• Lineweaver-Burke plot (double-reciprocal plot)

  Used to calculate Km and Vmax as well as to determine the mechanism of action of enzyme inhibitors

  
• 1/Vo = Km/Vmax(S) + 1/Vmax
• The intercept on the x axis is equal to -1/Km, the intercept on the y axis is equal to 1/Vmax
• Inhibitor


  Substance that can diminish the velocity of an enzyme-catalyzed reaction
• Types of inhibitors


  • Reversible inhibitors
  • Irreversible inhibitors
• Reversible inhibitors


  Bind to enzymes through noncovalent bonds, dilution of the enzyme-inhibitor complex results in dissociation of the reversibly-bounded inhibitor and recovery of enzyme activity
• Irreversible inhibitors


  The enzyme-inhibitor complex cannot be diluted, they act by forming covalent bonds with specific groups of enzymes
• Competitive inhibition


  Occurs when the inhibitor binds reversibly to the same site that the substrate would normally occupy and competes with the substrate for the site
• Effects of competitive inhibition

  • Effect on Vmax- reversed by increasing (S)

  2. Effect on Km- increases the apparent Km for a given substrate, more substrate is needed to achieve 1/2 Vmax

  3. Effect on Lineweaver-Burke plot- shows a characteristic plot in which the plots of the inhibited and uninhibited reactions intersect on the Y axis at 1/Vmax (Vmax is unchanged), the inhibited and uninhibited reactions show different X axis intercepts, indicating that the Km is increased