Enzymes

Enzymes: Catalysis, Mechanisms, and Regulation

Catalysts and Enzymes

• Enzymes are biological catalysts that accelerate chemical reaction rates.
• Catalysts increase reaction rates, enabling reactions that would otherwise be too slow or require higher temperatures.
• Proteins are effective catalysts due to their ability to bind a wide range of molecules.
• Enzymes bring reactants (substrates) together in the correct orientation for bond formation or breakage.
• Some enzymes require non-protein prosthetic groups, often derived from vitamins.
• Ribozymes are RNA molecules that catalyze reactions involving nucleic acids.

Reversibility and Physiological Conditions

• Chemical reactions are theoretically reversible.
• Under physiological conditions, many reactions are practically irreversible due to reactant and product concentrations.
• Enzymes catalyze both forward and reverse reactions.
• Cellular enzymes operate under physiological conditions: low temperature, aqueous environment, neutral pH.
• Enzymes accelerate reactions by factors of 10^6 to 10^{12} and are highly specific for their substrates.

Regulation of Enzyme Activity

• Enzyme activity is highly regulated to meet a cell's specific needs.
• Regulation mechanisms include:
  • Hormonal signals influencing enzymatic activities in other cells.
  • Proper folding into tertiary and quaternary structures to bring together specific amino acids for catalysis.

Active Site

• The active site is where the substrate binds to the enzyme.
• Substrates bind to enzymes via multiple weak, non-covalent interactions, forming an enzyme-substrate complex

Classes of Enzymes

• Proteases:
  • Split proteins by adding water across the peptide bond (hydrolysis).
  • Hydrolysis breaks the peptide bond, releasing a free carboxyl group and a free amino group.
• Hydrolyases:
  • A broader class of enzymes that break covalent bonds by adding water.
  • Examples: amylase (hydrolyzes starch), lipases (hydrolyze fatty acids of triacylglycerols).
• Dehydrogenases:
  • Oxidize substrates by removing hydrogen atoms. Lactate dehydrogenase converts lactate to pyruvate by removing two hydrogen atoms (two protons plus two electrons).
  • The hydrogen atoms are transferred to NAD^+, converting it to NADH + H^+.
  • The reaction is readily reversible.
  • Dehydrogenases are a subtype of oxidoreductases.
• Oxidoreductases:
  • Catalyze a broad variety of oxidation-reduction reactions.
  • The oxidation catalyzed by dehydrogenases does not require molecular oxygen.
• Other major classes: transferases, lyases, isomerases, and ligases.

Enzyme Activity Measurement

• Enzyme activity can be measured by changes in light absorption.
• Dehydrogenase activity is measured by changes in light absorption due to different spectra of NAD^+ and NADH.
• The rate of lactate dehydrogenase reaction can be monitored by measuring changes in optical density at 340 nm.

Enzyme Specificity

• Within a class of enzymes (e.g., proteases), individual enzymes are specific for different substrates.
• All proteases hydrolyze peptide bonds, but different proteases hydrolyze different bonds in the same or different proteins.
• Examples:
  • Trypsin:
    • Synthesized in the pancreas, contributing to intestinal hydrolysis of dietary protein.
    • Cleaves peptide bonds within proteins (endopeptidase).
    • Binds to the carboxyl side of basic amino acid residues (lysine and arginine).
  • Thrombin:
    • A plasma protein contributing to blood clotting.
    • Hydrolyzes arginine-glycine bonds only within specific peptide sequences.
  • Chymotrypsin:
    • Digestive protease produced by the pancreas.
    • Cleaves peptide bonds on the carboxy-terminal side of large hydrophobic amino acids (e.g., tyrosine, tryptophan, phenylalanine, and methionine).
    • The enzyme becomes temporarily covalently modified during the process.

Catalytic Mechanism of Chymotrypsin

• Peptide bonds are extremely stable in the absence of catalysis.

• Chymotrypsin catalyzes proteolysis in two steps:

  • Acylation:
    • Splits the amide bond, releasing the newly freed amino group.
    • The carboxyl group of the amide bond remains covalently bonded to the hydroxyl group of an active serine residue.
  • Deacylation:
    • Hydrolysis of the bond holding the carboxyl group to the enzyme.
    • Releases the carboxyl group and its attached portion of the original polypeptide from the enzyme.

• Catalytic Triad:

  • The active site of chymotrypsin involves three amino acid residues: histidine, aspartate, and serine (Serine 195).
  • Interactions within the triad result in the removal of a hydrogen atom from the hydroxyl group of serine, creating a highly active alkoxide ion.
  • The alkoxide ion can break the peptide bond of the substrate and form a carboxyl-enzyme intermediate.

• Serine Protease Family:

  • Serine proteases share the same catalytic strategy of using a histidine and aspartate to activate serine.
  • Includes chymotrypsin-related digestive enzymes like trypsin and elastase (40% identical amino acid sequences).
  • Also includes human serine proteases involved in blood clotting.
  • These proteases have similar catalytic mechanisms but different substrate specificities.

Substrate Specificity

• Serine proteases share a catalytic triad (serine, histidine, aspartate) but differ in substrate specificity.
• Chymotrypsin has a hydrophobic pocket adjacent to Serine 195 that binds large hydrophobic or aromatic amino acid side chains (tyrosine, tryptophan, phenylalanine, methionine).
• Trypsin has a carboxyl group (aspartate) in the pocket, allowing stable association of positively charged amino acid side chains (arginine and lysine).
• Elastase has a smaller binding pocket due to bulky side chains, favoring hydrolysis of amino acids with small side chains (glycine and alanine).

Different Classes of Proteases

• Serine proteases: Use a serine in the active site as a nucleophile.
• Cysteine proteases: Use a cysteine in their active site (e.g., caspases).
• Aspartyl proteases: Use an aspartate in the active site (e.g., renin, HIV proteases).
• Metalloproteases: Contain a divalent cation (usually zinc) in the active site (e.g., matrix metalloproteases).

Enzymes and Activation Energy

• Enzymes lower the activation energy required for a reaction by increasing the probability a particular reaction will occur.
• Reactions still require an input of energy to occur.
• Enzymes reduce the activation energy without altering the net free energy change of the reaction.

Exergonic Reactions

• Some enzyme-catalyzed reactions are exergonic (release energy).
• The free energy change (\Delta G) is negative.
• The energy level of the product is lower than that of the substrate.
• Formation of the activated intermediate (transition state) requires net energy input.

Transition State and Equilibrium

• The highest energy intermediate is the transition state.
• Enzymes catalyze both forward and reverse reactions.
• The net effect is an equilibrium between the two reactions.
• The relative rates of the two reactions depend on the difference in \Delta G between reactants and products.

Factors Affecting Reaction Direction

• The free energy change \Delta G of any reaction depends on the actual concentrations of substrates and products.
• Depending on specific conditions, a reaction can run forward or backward.
• Some reactions (e.g., lactate dehydrogenase) are readily reversible, depending on concentrations of lactate, NAD^+, pyruvate, and NADH.
• Enzymes increase the rate of the reaction but do not alter the equilibrium.
• Reactions are essentially irreversible under physiological conditions when the equilibrium completely favors one set of products or when one of the products is rapidly removed.

Endergonic Reactions

• Endergonic reactions have a \Delta G > 0 and require a net input of energy.
• Endergonic reactions occur at a significant rate only when coupled to other reactions that provide energy (exergonic reactions).