Biochem Sept. 22nd
Enzymes: Catalysis, Mechanisms, and Specificity
Introduction to Enzymes
Biological Catalysts: Enzymes are biological catalysts that accelerate or regulate most chemical transformations within cells.
Unchanged by Reaction: Enzymes participate in chemical reactions but are not permanently altered themselves; they are regenerated at the end of the reaction.
Composition: Most enzymes are proteins, though a small number are RNA (ribozymes).
Nomenclature: Enzyme names often end with "-ase" (e.g., reductase, amylase). Some are named after the reaction they catalyze, leading to potentially long, descriptive names.
Substrates: Molecules upon which an enzyme acts, converting them into products.
Key Characteristics of Enzymes
Molecular Machines: Enzymes are highly efficient molecular machines essential for life, turning reactions that might take years into seconds or faster.
High Specificity: Enzymes typically recognize only one or a few specific substrates out of thousands of molecules in a cell, ensuring precise reactions.
High Accuracy: Enzyme catalysis is usually very accurate, reliably carrying out specific biochemical tasks.
Reaction Acceleration: Enzymes can accelerate reaction rates by billions or even trillions of times (10^9 to 10^{12} times) compared to uncatalyzed reactions.
Example 1: Carbonic Anhydrase: Catalyzes \text{CO}_2 hydration, increasing the rate by approximately 10^7 times.
Example 2: Chymotrypsin: Catalyzes peptide bond hydrolysis. Without the enzyme, it would take approximately 20 years to hydrolyze half of the peptide bonds. With chymotrypsin, the catalytic rate is about 190 per second, representing an increase in the order of trillions.
Mild Conditions: Unlike many chemical catalysts, enzymes function effectively under mild biological conditions (e.g., \text{37}^{\circ}\text{C} body temperature, normal pressure, neutral pH).
Enzyme Classification
Enzymes are classified by the type of reaction they catalyze:
Oxidoreductases: Oxidation-reduction reactions.
Transferases: Transfer of functional groups (e.g., kinases transferring a phosphate group from ATP).
Hydrolases: Hydrolysis reactions.
Others include lyases, isomerases, and ligases.
Active Site and Enzyme-Substrate Complex
Active Site: A small, specific part of the enzyme's surface where the substrate binds and the chemical reaction occurs. It's often a pocket or cleft between domains.
Substrate Binding: Substrates bind to the active site via weak non-covalent interactions (similar to protein-ligand binding, but leading to reaction).
Reaction Pathway: Substrate (S) binds to Enzyme (E) to form an Enzyme-Substrate complex (ES). The ES complex converts to an Enzyme-Product complex (EP), from which the Product (P) is released, regenerating the free Enzyme (E).
\text{E} + \text{S} \rightleftharpoons \text{ES} \longrightarrow \text{EP} \rightleftharpoons \text{E} + \text{P}
Enzyme Kinetics and Thermodynamics
Spontaneous Reactions: A reaction is spontaneous if the free energy of the products is lower than that of the reactants (\Delta G \text{ is negative}).
Enzymes and Equilibrium: Enzymes do not change the equilibrium position of a reaction or the overall standard free energy change (\Delta G) between reactants and products. They only affect the rate at which equilibrium is reached.
Transition State: An unstable, high-energy intermediate form that reactants must pass through to become products. It represents the point of highest free energy along the reaction pathway, where bonds are in the process of breaking and forming.
Activation Free Energy (\Delta G^{\ddagger}): The free energy difference between the transition state and the reactants. A higher \Delta G^{\ddagger} means a slower reaction rate.
Enzyme Mechanism: Enzymes accelerate reactions by lowering the \Delta G^{\ddagger}, making it easier and faster for reactants to reach the transition state.
The difference in activation free energy between an uncatalyzed and catalyzed reaction is denoted as \Delta \Delta G^{\ddagger}.
Strategies for Lowering Activation Energy
Proximity and Orientation: Enzymes act as a platform, bringing substrates together in the correct orientation to facilitate bond formation, increasing the effective concentration of reactants.
Charge Stabilization: Active site residues can use their own charges to influence the charge distribution of the substrate, making bond breaking or formation easier.
Stabilization of Transition State: Enzymes preferentially bind to and stabilize the transition state, effectively lowering its free energy and thus reducing the overall \Delta G^{\ddagger} without affecting the stability of initial reactants or final products.
Transition State Analogs: Molecules that resemble the transition state but cannot be converted into product. They bind to the enzyme with very high affinity, often more tightly than the substrate itself, making them potent enzyme inhibitors. This is a strategy for drug design.
Cofactors and Coenzymes
Cofactors: Non-protein partners required by some enzymes for function.
Metal Ions: Simple inorganic ions (e.g., iron, zinc) that bind to the enzyme.
Coenzymes: Organic molecules that act as cofactors.
Co-substrates: Coenzymes that bind transiently to the enzyme, often undergoing modification (e.g., oxidation/reduction) and then dissociating.
Prosthetic Groups: Coenzymes that are permanently (tightly and stably) bound to the enzyme, similar to heme in myoglobin/hemoglobin.
Chymotrypsin: An Example of Enzyme Catalysis
Type: A member of the serine protease family, characterized by a critical serine residue in its active site.
Source and Function: Secreted by the pancreas into the intestine to break down dietary proteins through hydrolysis of peptide bonds.
Reaction Type: Hydrolysis – adding a water molecule to break a peptide bond.
Catalytic Triad: Three critical amino acid residues in the active site work together:
Serine 195 (Ser195): The nucleophile that attacks the carbonyl carbon of the peptide bond.
Histidine 57 (His57): Acts as a general acid-base catalyst, abstracting a proton from Ser195 to increase its nucleophilicity, and later donating a proton to stabilize the leaving group (amine).
Aspartic Acid 102 (Asp102): Stabilizes the positively charged His57 through a strong hydrogen bond, increasing its basicity and ability to abstract protons.
Note: The specific residue numbers are not to be memorized, but their roles are crucial.
Chymotrypsin Mechanism (Hydrolysis of a Peptide Bond)
Goal: Cleave the amide bond (scissor bond) in the substrate (R\text{N-CO}-R\text{C}, where R\text{N} is the N-terminal side and R\text{C} is the C-terminal side).
Part 1: Formation of Acyl-Enzyme Intermediate and Release of C-terminal Product
Substrate Binding: The substrate binds to the active site, positioning the scissile peptide bond (e.g., between residue 10 and 11) close to Ser195.
Nucleophilic Attack (Step 1):
His57 abstracts a proton from the hydroxyl group of Ser195.
This makes Ser195 a highly nucleophilic alkoxide ion.
Ser195's oxygen attacks the electrophilic carbonyl carbon of the peptide bond.
The carbon-oxygen double bond breaks, and electrons move to form a negatively charged oxygen.
First Tetrahedral Intermediate (Transition State 1):
A transient, high-energy tetrahedral intermediate is formed, with a negatively charged oxygen (oxyanion).
This intermediate is stabilized by the positive charge on His57 (stabilized by Asp102) and by interaction with the oxyanion hole.
Proton Transfer & Cleavage (Step 2):
His57, now positively charged, donates the abstracted proton to the amide nitrogen of the peptide bond (the leaving group).
The carbon-nitrogen bond breaks, releasing the C-terminal portion of the peptide (R\text{C}) as the first product.
The acyl group (R\text{N-CO}-) remains covalently attached to Ser195.
Acyl-Enzyme Intermediate: A stable intermediate where the N-terminal part of the substrate is covalently linked to the enzyme's Ser195.
Part 2: Deacylation and Release of N-terminal Product (Reversing Part 1 with Water)
Water Entry: A water molecule (\text{H}_2\text{O}) enters the active site and binds in a position analogous to the original substrate's serine-attacking oxygen.
Nucleophilic Attack (Step 3):
His57 (now neutral again) abstracts a proton from the water molecule.
This makes the water molecule's oxygen a strong nucleophile.
The nucleophilic oxygen attacks the carbonyl carbon of the acyl-enzyme intermediate.
The carbon-oxygen double bond reforms, and electrons move, forming a negatively charged oxygen.
Second Tetrahedral Intermediate (Transition State 2):
A second transient, high-energy tetrahedral oxyanion intermediate is formed.
This intermediate is stabilized by the positive charge on His57 (stabilized by Asp102) and interactions within the oxyanion hole.
Proton Transfer & Enzyme Regeneration (Step 4):
His57 donates the proton back to the Ser195 oxygen.
The covalent bond between Ser195 and the N-terminal acyl group breaks.
The N-terminal part of the original substrate (R\text{N-COOH}) is released as the second product.
Enzyme Regeneration: The enzyme is regenerated in its original active form, ready for another catalytic cycle.
Reaction Coordinate Diagram (Conceptual)
Starts at Reactants (higher energy).
Rises to Transition State 1 (First Tetrahedral Intermediate).
Drops to a local minimum at the Acyl-Enzyme Intermediate.
Rises again to Transition State 2 (Second Tetrahedral Intermediate).
Finally drops to Products (lower energy than reactants for a spontaneous reaction).
Role of the Oxyanion Hole
Specific Structure: A pocket formed by amide hydrogens of Ser195 and Gly193 (close to Ser195).
Transition State Stabilization: In the planar ground state, the carbonyl oxygen of the substrate does not interact significantly with the oxyanion hole. However, once the substrate's carbonyl carbon undergoes nucleophilic attack and becomes tetrahedral, the oxygen becomes negatively charged (oxyanion) and fits into the oxyanion hole.
Hydrogen Bonding: The oxyanion forms strong hydrogen bonds with the amide hydrogens of Ser195 and Gly193.
Energetic Contribution: These strong hydrogen bonds provide significant stabilization to both tetrahedral transition states, further lowering the \Delta G^{\ddagger} and increasing the reaction rate.
Enzyme Specificity: The Specificity Pocket
Serine proteases, despite having similar active sites, exhibit different substrate specificities due to variations in their specificity pocket (often called the S1 pocket, adjacent to the active site).
Chymotrypsin Specificity: Preferentially cleaves peptide bonds where the N-terminal residue of the scissile bond has a large hydrophobic side chain (e.g., phenylalanine, tyrosine, tryptophan, methionine).
Its specificity pocket is a large, hydrophobic cavity lined with small glycine residues, which readily accommodates bulky hydrophobic side chains.
Binding of the large hydrophobic residue in this pocket correctly positions the scissile bond in the active site for catalysis.
Trypsin Specificity: Preferentially cleaves peptide bonds where the N-terminal residue of the scissile bond is a positively charged residue (e.g., lysine, arginine).
Its specificity pocket contains a negatively charged aspartate residue (Asp189) at its base, which attracts and forms a salt bridge with positive charges.
Elastase Specificity: Preferentially cleaves peptide bonds where the N-terminal residue of the scissile bond is a small, uncharged residue (e.g., alanine, glycine).
Its specificity pocket is largely blocked by bulky valine and threonine residues, allowing only small side chains to fit. This demonstrates how enzyme structure dictates substrate binding and catalytic efficiency for specific substrates.