Biochemistry Chapter on Enzyme Catalysis

Local Environment Influence on pKa

• Glutamate sidechains typically have a pKa of 4.1.
• Lysozyme activity spans a pH range of ~4 to 9.
• Human tears have an average pH of ~7.4.
• pH > pKa leads to deprotonation of the molecule in question.
• pH < pKa leads to protonation of the molecule in question.
• At the optimal pH for lysozyme (5), Glu sidechains would be negatively charged.
• Local environment critically influences sidechain pKa values.

Enzyme Catalysis

• Enzyme-catalyzed reactions occur in active sites.
• Enzymes bind and position substrates to facilitate reactions in enzyme-substrate complexes.
• Maximum reaction velocity is reached when all active sites are filled with substrate.

Rate Enhancement Mechanisms

• Enzymes increase reaction rates through proximity and orientation effects, bringing substrates closer together and arranging them optimally.
• Most rate enhancement originates from binding, transforming intermolecular reactions into faster intramolecular ones.
• Substrates are brought closer together and oriented optimally for reaction.
• This is exemplified in a reaction where negatively charged oxygen is positioned correctly near a target carbon. Without enzymes, reactants collide randomly. With enzymes, reactants are linked to catalyse the reaction, enhancing reaction speed. Linking progresses from a CH2 group, to direct linkage, to fixed linkage to yield increasing speed.

Transition States and Intermediates

• Reactions proceed through transition states and sometimes intermediates from substrate to product.
• Transition states exist briefly, in femtosecond timescales.
• Intermediates are more stable, potentially isolable.

Transition State Affinity

• Enzymes bind transition states, lowering activation barriers.
• Transition state analogues are excellent inhibitors and are used in catalytic antibody design., better than substrates in some cases, like proline racemase.
• Proline racemase's transition state analogues bind significantly better than the proline substrate.

Enzyme Substrate Binding

• Tight substrate binding is detrimental because it elevates the activation energy barrier.
• Catalytic enzymes typically have milli- to micromolar substrate binding constants.
• Binding proteins/antibodies have nano- to picomolar constants (stronger binding).

Enzyme Structure and Function

• Enzymes are unchanged throughout the reaction cycle, even with covalent intermediates, which are later broken.
• High substrate specificity, matching active site shape and functional groups.
• Lock-and-key model is not representative of all enzyme interactions.

Flexibility of Enzymes

• Enzyme structures are flexible, adapting to optimal substrate/transition state binding.
• Induced fit model describes this flexibility.
• Strain energy in the enzyme is relieved during the reaction, stabilizing the transition state and accelerating the reaction.

Catalytic Mechanisms

• Catalysis accelerates the approach to equilibrium.
• Most rate enhancement is from substrate binding/orientation; following common mechanisms:
  • General acid-base catalysis
  • Covalent catalysis
  • Metal ion catalysis

General Acid-Base Catalysis

• Enzymes act as acids (proton donors) or bases (proton acceptors) affecting reaction rates.
• Lysozyme (example): Glu35 acts as an acid and a base, utilizing water addition in its reaction mechanism. Glu35 donates a proton to the substrate, facilitating the cleavage of the bond. Then Glu35 extracts a proton from a water molecule to complete the reaction.

Covalent Catalysis

• Many enzymes form covalent bonds with substrates, generating transient reactive intermediates.
• Lysozyme uses a covalent intermediate between Asp52 and the oxonium ion on the NAM sugar during its catalytic cycle.. A water molecule attacks the carbon in the NAM ring, breaking the bond to Asp52.

Metal Ion Catalysis

• Enzymes often use bound metal ions in their reaction mechanisms.
• Metal ions can be tightly bound (metalloenzymes) or loosely associated (metal-activated).
• Metals can generate nucleophilic species for catalysis or stabilize the transition state or increase binding interactions.
• Examples include carbonic anhydrase, DNA polymerase I, and Fe-S clusters.

Cofactors and Coenzymes

• Enzymes require cofactors (metal ions or small molecules) for catalytic activity.
• Cofactors are recycled in the cell.
• Coenzymes are often small organic molecules; tightly bound ones are prosthetic groups.
• Loosely bound/dissociable ones are cosubstrates.
• Apoenzyme + Cofactor = Holoenzyme.
• Examples of cofactors include AMP, ATP, NAD, and NADP.
• NAD(P)+/NAD(P)H are freely dissociable and act as important sources of reducing power.
• The nicotinamide ring is accessible in the enzyme active site.

Enzyme Classification

• Enzymes are grouped based on the type of reaction they catalyze:
  • Oxidoreductases (dehydrogenases): redox reactions, often using NAD(P)H, FAD, or iron-sulfur clusters.
  • Transferases: transfer groups between molecules.
  • Hydrolases: cleavage reactions using water.
  • Lyases (synthases): addition or removal of groups to form double bonds.
  • Isomerases: interconversion of isomers.
  • Ligases (synthases): joining of molecules, often requiring ATP.