Biochemistry Chapter 9 : Enzymatic Catalysis

Explain why enzymes are so special

1) Extremely fast reaction rates

• Without enzyme → 0.13 reactions/sec

• With enzyme → 1,000,000 reactions/sec

• Enzymes can increase reaction rates millions of times faster

2) High substrate specificity

• Enzymes usually bind:

  • one specific molecule, or

  • a narrow range of similar molecules

• The specificity occurs b/c of:

  • have to be complementary by shape ( geometric) and charge (electronic) between the enzyme active site and substrate

3) Conformational flexibility

• Enzymes change shape when the substrate binds

• Allows:

  • optimal positioning of substrates

  • efficient catalysis

• After reaction:

  • products leave

  • the enzyme returns to its original shape

4) Lower activation energy

• Enzymes speed reactions by lowering the activation energy needed to reach the transition state, but they do not change the overall free energy (ΔG) of the reaction.

Define cofactors

• non-amino acid components required for enzyme activity

• Assist enzymes in performing chemical reactions

• Ex.) B vitamins involved in cellular respiration

Define coenzymes

• organic cofactors that help enzymes perform reactions

• They typically:

  • carry electrons

  • transfer chemical groups

  • assist metabolic reactions

Explain how to interpret transition state diagrams

• Reaction coordinate diagram shows how free energy changes as reactants become products

• ΔG:

  • Negative: Reaction is favorable (spontaneous) → Exergonic

  • Positive: Reaction is unfavorable (unspontaneous) → Endergonic

  • Zero: Reaction is at equilibrium

Define a Transition state and how it determines reaction speed

• The highest energy structure during a reaction and the most unstable structure along the reaction path

• Higher barrier/ peak → slower reaction

• Lower barrier/peak → faster reaction

Define Activation Energy

• The energy required to reach the transition state

• The barrier substrates must overcome to become products

What do enzymes change, and what don’t they change?

• Change:

  • Lower the activation energy

  • stabilize transition state

• Don’t Change:

  • ΔG of reaction

What are the 6 types of reactions used by enzymes

• Oxidoreductase

• Transferase

• Hydrolase

• Lyase

• Isomerase

• Ligase

Oxidoreductase

• Catalyze oxidation-reduction reactions

• anything with NAD+, NADH+H+, or FAD

Transferase

• Transfer functional groups from one molecule to another

• Ex.) Kinase

Hydrolase

• Break bonds using water (Hydrolysis)

• Ex.) RNase A and lysozyme

Lyase

• Break bonds (elimination) to form double bonds or rings

Isomerase

• Rearrangement within a molecule with same number of carbons

• Catalyze isomerization reactions

Ligase

• Bond formation coupled using ATP hydrolysis

What are the 5 ways enzymes lower activation energy?

• Acid-Base Catalysis

• Covalent Catalysis

• Metal Ion Catalysis

• Proximity and Orientation Effects

• Preferential Binding of the Transition State

Acid-Base Catalysis

• Movement of H+ (proton transfer) stabilizes the transition state and makes it more favorable for a reaction

• Ex.) Histidine residues acting as acids or bases

Covalent Catalysis

• Enzyme forms a temporary covalent bond with the substrate, creating an intermediate that speeds the reaction

Metal Ion Catalysis

• Metal ions:

  • help orient substrates

  • stabilize charged intermediates

  • activate water molecules for hydrolysis

• Ex) Zn2+ in carbonic anhydrase

Proximity and Orientation Effects

• The enzyme active site:

  • Brings substrates close together

  • Aligns them in the correct orientation for reaction

Preferential Binding of the Transition State

• Enzymes bind the transition state more tightly than the substrate or product, stabilizing the highest-energy structure.

Explain the utility (usefulness) of transition state analogs

• Transition state analogs: mimic the structure of the transition state

• Because enzymes bind the transition state most strongly:

  • these molecules bind very tightly

  • they can block enzyme activity

• Utility:

  • enzyme inhibitors

  • tools to study enzyme mechanisms

  • There effectiveness comes from the enzyme’s preferential binding to the transition state

Explain why serine proteases are a protein family

• Protein family: proteins grouped together based on similar structure and/ or function

• Why:

  • perform peptide bond hydrolysis

  • share a similar catalytic mechanism

  • have similar 3D active sites

• But:

  • primary amino acid sequences may differ

• Examples:

  • digestive enzymes: chymotrypsin, trypsin, elastase

Describe the Specificity of Serine Proteases binding pocket:

• Binding pocket: determines the substrate specificity of the various serine proteases

• Chymotrypsin:

  • pocket is a deep hydrophobic pocket that allow long, uncharged amino acids like F (phenylalanine) and W (tryptophan) to fit in chymotrypsin

• Trypsin:

  • has a negative charged pocket, so better for K (lysine) and R (arginine) amino acids

• Elastase:

  • has a nonpolar pocket but w/ bigger R-groups protruding into it, therefore it does smaller nonpolar amino acid like A (alanine)

Define the role of the catalytic triad in serine proteases

• Catalytic triad consists of three amino acids:

  • Serine:

    • nucleophile that attacks peptide bond

  • Histidine:

    • acts as proton donor/ acceptor

  • Aspartate:

    • stabilizes histidine

• These residues work together to perform catalysis

• Although they act together in the active site:

  • they are not located near each other in the primary sequence

  • protein folding brings them together in the active site

What are the key steps of catalysis for serine proteases?

1) Activation of serine

• Histidine removes a hydrogen from Ser195, creating a strong nucleophile

2) Nucleophilic attack

• Ser195 attacks the carbonyl carbon of the peptide bond, producing a tetrahedral intermediate

3) Peptide bond cleavage

• The unstable intermediate collapses:

  • peptide bond breaks

  • one product leaves

  • the other remains attached to Ser195

4) Water enters active site

• Water is positioned to attack the enzyme-substrate intermediate

5) Hydrolysis

• Water attacks the intermediate, breaking the bond between the enzyme and substrate

6) Enzyme regeneration

• The second product is released and the enzyme returns to its original state

Describe how allosteric effectors impact the structure of an enzyme

• Allosteric inhibition:

  • binds to different location of active site but causes a change in conformation

  • changes shape of active site, so substrate can no longer bind

• Allosteric activation:

  • binds to a site on the enzyme other than the active site and increases the enzyme’s activity (catalytic activity)

Enzyme lower the energy of a transition state by:

• Proper orientation:

  • small spot relative to the total physical space of enzyme

  • usually occur in clefts and crevices in the protein

• Functional groups:

  • key groups bind to substrate and catalyze its conversion to products

• Coupling:

  • Thermodynamically unfavorable reactions can be driven by coupling them to thermodynamically favorable reactions