Enzymes, Enzymatic Reactions, Mechanism, and Regulation

Enzymes as Catalysts

• Enzymes are biological catalysts that accelerate chemical reaction rates.
• They enable reactions to proceed at physiological temperatures by overcoming high activation energy barriers.
• Proteins are effective catalysts due to their ability to bind a wide range of molecules.
• Enzymes bring reactants (substrates) together in specific orientations to facilitate 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, but many are practically irreversible under physiological conditions due to reactant/product concentrations.
• Enzymes catalyze both forward and reverse reactions.
• Cellular enzymes operate under physiological conditions: low temperature, aqueous environment, and neutral pH.
• They can accelerate reactions by factors of $10^6$ to $10^{12}$ and are highly specific for their substrates.

Regulation of Enzyme Activity

• Enzyme activity is tightly regulated to meet specific cellular needs.
• Hormonal signals allow cells to influence enzymatic activities in other cells.
• Proper protein folding into tertiary and quaternary structures is crucial for enzyme function.
• Specific amino acids from different parts of the polypeptide chain (e.g., A, B, C) come together to form the active site and contribute to catalysis.

Active Site

• The active site is the region on an enzyme where the substrate binds and undergoes a chemical reaction.
• It's a three-dimensional cleft formed by specific amino acid residues (e.g., A, B, C).
• Substrates bind to enzymes via multiple weak, non-covalent interactions, forming an enzyme-substrate complex.

Enzyme Classes

• Proteases: Enzymes that hydrolyze peptide bonds between amino acids in proteins.
  • Water is added across the peptide bond, breaking it into a free carboxy group and a free amino group.
  • Example: Chymotrypsin, trypsin, elastase.
• Hydrolyases: A broader category that includes proteases; they break covalent bonds by adding water.
  • Examples: Amylase (hydrolyzes starch), Lipases (hydrolyzes fatty acids in triacylglycerols).
• Dehydrogenases: Oxidize substrates by removing hydrogen atoms.
  • Example: Lactate dehydrogenase converts lactate to pyruvate by removing two hydrogen atoms (2 protons + 2 electrons).
  • The hydrogen atoms are transferred to $NAD^+$, forming $NADH + H^+$.
  • The reaction is readily reversible.
• Oxidoreductases: Catalyze a broad range of oxidation-reduction reactions; dehydrogenases are a subtype.
  • The oxidation catalyzed by dehydrogenases does not require molecular oxygen.
• Transferases
• Lyases
• Isomerases
• Ligases

Measuring Enzyme Activity

• Enzyme activity can be measured by observing changes in light absorption.
• Dehydrogenase activity is readily measured this way since $NAD^+$ and $NADH$ have different UV light absorption spectra.
• The rate of the lactate dehydrogenase reaction can be monitored by measuring changes in optical density at 340 nm, where the differences are most pronounced, with NADH absorbing strongly.

Enzyme Specificity

• Within each enzyme class, individual enzymes exhibit specificity for different substrates.
• Proteases hydrolyze peptide bonds, but different proteases cleave different bonds in the same or different proteins.
  • Trypsin: Synthesized in the pancreas, it cleaves peptide bonds on the carboxyl side of basic amino acid residues like lysine and arginine (endopeptidase).
  • Thrombin: A plasma protein involved in blood clotting; it hydrolyzes arginine-glycine bonds only within specific peptide sequences.

Catalytic Mechanism: Chymotrypsin Example

• Chymotrypsin is a digestive protease produced by the pancreas, illustrating a well-understood catalytic mechanism.
• Peptide bonds are generally stable, but chymotrypsin accelerates their hydrolysis in the small intestine.
• Chymotrypsin has a reactive serine residue in its active site.
• It cleaves peptide bonds on the carboxy-terminal side of large hydrophobic amino acids (tyrosine, tryptophan, phenylalanine, methionine).
• The enzyme undergoes temporary covalent modification during the process.

Chymotrypsin Mechanism: Acylation and Deacylation

• Chymotrypsin-catalyzed proteolysis occurs in two steps:
  • Acylation: The amide bond is split, releasing the newly freed amino group and its attached polypeptide portion. The carboxyl group of the amide bond forms a covalent bond with the hydroxyl group of the active serine residue.
  • Deacylation: The bond holding the carboxyl group to the enzyme is hydrolyzed, releasing the carboxyl group and its attached polypeptide portion.

Catalytic Triad

• The active site of chymotrypsin contains a catalytic triad: serine, histidine, and aspartate.
• The interaction of these amino acids results in the removal of a hydrogen atom from the serine hydroxyl group, creating a highly reactive alkoxide ion.
• Serine 195 refers to the specific serine residue in the polypeptide chain that participates in the active site.
  • The alkoxide ion on serine 195 can break the peptide bond of the substrate, forming a carboxyl-enzyme intermediate.

Serine Proteases

• A family of serine proteases uses the same catalytic strategy: a histidine and aspartate activating a serine to form an active alkoxide ion.
  • This group includes trypsin and elastase, which are about 40% identical in amino acid sequence to chymotrypsin.
  • Another group is involved in blood clotting.
• Serine proteases share a catalytic mechanism, but differ in substrate specificity.

Substrate Specificity

• Serine proteases have a catalytic triad of serine, histidine, and aspartate.
• Chymotrypsin has a hydrophobic pocket adjacent to serine 195 that accommodates large hydrophobic amino acid side chains (tyrosine, tryptophan, phenylalanine, methionine).
• Trypsin has an aspartate in the pocket, which attracts positively charged amino acids like arginine and lysine.
• Elastase has a smaller pocket due to bulky amino acids, so it binds to amino acids with small side chains like glycine and alanine.

Protease Classes

• Serine proteases use a catalytic triad with serine as the active nucleophile.
• Cysteine proteases have a cysteine in the active site (e.g., caspases).
• Aspartyl proteases have an aspartate in the active site (e.g., renin, HIV protease).
• Metallo proteases contain a divalent cation (usually zinc) in the active site (e.g., matrix metalloproteases).

Enzymes Lower Activation Energy

• Enzymes decrease the activation energy required for a reaction.
• Energetically favorable reactions still require energy input to occur.
• Enzymes increase the probability of a reaction by lowering the activation energy.

Exergonic Reactions

• Some enzyme-catalyzed reactions are exergonic (release energy).
• Burning wood is an example of a non-enzymatic exergonic reaction.
• In exergonic reactions, the energy level of the product is lower than that of the substrate (negative $\Delta G$).
• Activation energy is still needed to reach the transition state.
• Enzymes lower the activation energy but do not change the net free energy change of the reaction.
• The enzyme lowers the "bump," but the reaction still proceeds from the same starting level to the same lower level.

Transition State

• In complex reactions with multiple reactants and products, the highest energy intermediate is called the transition state.
• Enzymes stabilize the transition state, decreasing the required activation energy.

Reversible Reactions and Equilibrium

• An enzyme that catalyzes a forward reaction also catalyzes the reverse reaction.
• The net effect is an equilibrium between the forward and reverse reactions.
• The relative rates depend on the difference in $\Delta G$ between reactants and products.
• When there is little difference in height on the two sides of the "hump," equilibrium is achieved.
• The free energy change ($\Delta G$) depends on substrate and product concentrations.
• Depending on conditions, a reaction can run forward or backward.
• Enzymes increase the reaction rate but do not alter the equilibrium.

Irreversible Reactions and Endergonic Reactions

• Some reactions are essentially irreversible under physiological conditions due to equilibrium favoring one side or rapid product removal.
• Endergonic reactions (\Delta G > 0) require net energy input.
• Endergonic reactions occur at a significant rate when coupled to exergonic reactions.