chapter 8

Catalytic Strategies Used by Enzymes

• Enzymes catalyze reactions by employing one or more strategies.
• Covalent catalysis utilizes a reactive group, usually a nucleophile in the active site.
  • The active site is temporarily modified during the reaction
  • The region is attracted to a positive charge in a molecule
  • The molecule donates electrons to an electron deficient chemical
• General acid-base catalysis involves molecules other than water that act as proton donors or acceptors.
• Metal ion catalysis can make a metal ion serve as an electrophilic catalyst and stabilizes negative charge on a reaction intermediate.
  • It can generate a nucleophile by increasing the acidity of a nearby molecule like water.
  • It can bind to the substrate, increasing interactions with the enzyme an dthe binding energy, and the enzyme often required cofactors.
• Catalysis by approximation and orientation involves reactions with two distinct substrates that are brought into close proximity and specific orientation on a single binding surface of the enzyme.

Enzyme Activity Modulation

• Temperature, pH, and inhibitory molecules can modulate enzyme activity.
• Increased temperature usually results in an increased rate of reactions, due to increased Brownian motion, increasing the chances of interactions between the enzyme and substrate.
• Enzymes denature above a certain temperature, stopping catalytic activity, as weak bonds holding the 3D structure together are broken.
  • Endotherms aren't affected by changes in body temperature
  • Ectotherms are more sensitive to changes in temperature
  • Thermophilic archaea have adapted to high temperatures without denaturing proteins
• Most enzymes have an optimal pH at which the enzyme has maximal acitivity, that varies with the enzyme type and is correlated to the environment of the enzyme. Active sites can be dependent on pH of the solution.
  • If enzyme requires ionization of glutamic acid and lysine residues at active site, then pH should be around neutral
  • -Coo- and -NH3+ groups leave the enzyme inactive

Enzyme Inhibition

• Enzyme inhibition is reversible or irreversible.
• Reversible inhibition involves rapid dissociation of the enzyme-inhibitor complex.
  • There are three types: competitive, uncompetitive, and noncompetitive.
• Irreversible inhibition means the inhibitor dissociates slowly from the enzyme and binds tightly either by covalent or noncovalent bonds.

Reversible Inhibitors

• Competitive inhibition has an inhibitor that is similar in structure to the substrate and binds to the active site, preventing binding of the regular substrate.
  • The rate of catalysis is decreased by reducing the amount of enzyme bound to the substrate but is overcome by adding more substrate, where it outcompetes the inhibitor for the active site.
• Uncompetitive inhibition occurs when the inhibitor binds only to the enzyme-substrate complex, and is substrate-dependent.
  • The binding site for the inhibitor is only present when the substrate is bound to the enzyme and adding more substrate cannot relieve the inhibition.
• Noncompetitive inhibition occurs when the inhibitor and substrate can bind the enzyme at the same time at different sites.
  • The inhibitor acts to decrease the overall number of active enzymes rather than decreasing the amount of enzyme bound to the substrate.
  • Changing substrate concentration cannot relieve inhibition.

Kinetics of Reversible Inhibitors

• Reversible inhibitors can be distinguished through kinetics.
• Competitive inhibition is when an increase in substrate concentration overcomes inhibition, Vmax will stay the same with and without inhibitor, increases apparent value of KM, more substrate is needed to obtain the same reaction rate or Vmax.
• Uncompetitive inhibition is when the enzyme substrate inhibitor complex does not produce product, Vmax is always lower in presence of inhibitor, reduction in apparent value of KM.
• Noncompetitive inhibition is when enzyme substrate inhibitor complex does not produce product, Vmax is lower in presence of an inhibitor and the KM is unchanged

Irreversible Inhibitors

• Four categories of irreversible inhibitors including:
  • Group-specific reagents - modifies specific R groups of amino acids.
    • Diisopropylphosphofluoridate (DIPF) inhibits enzymes by covalently modifying specific serine residue within active site of enzyme.
  • Affinity labels (substrate analogs) - covalently modifies active-site residues.
    • Structurally similar to an enzyme's substrate and is more specific for enzyme active site than group-specific reagents.
  • Suicide inhibitors - chemically modified substrates that bind to an enzyme.
    • Processed by normal catalytic mechanism, reactive intermediate is formed that inactivates enzyme by covalent modification, modified group of enzyme is vital to enzyme function.
  • Transition-state analogs - potent inhibitors of enzymes.
    • Bind tighter to enzyme to prevent binding of substrate.