Enzymatic Catalysis Notes

9.1: General Properties of Enzymes

• Enzymes are primarily proteins and are categorized into seven mechanistic classes.
• They enhance reaction rates by a factor of at least $10$.
• Substrate specificity depends on the active site's geometric and electronic characteristics.
• Enzymes differ from ordinary catalysts:
  • Higher reaction rates: Enzymatic reactions are significantly faster.
  • Milder reaction conditions: Reactions occur at temperatures below $100°C$, atmospheric pressure, and neutral pH.
  • Greater reaction specificity: Enzymatic reactions produce minimal side products.
  • Capacity for regulation: Enzyme activity is controlled by substance concentrations through mechanisms like allosteric control, covalent modification, and varying enzyme synthesis.

Enzymes Increase Reaction Rate

• Table 9.1 provides a comparison of nonenzymatic and enzymatic reaction rates, highlighting the rate enhancement achieved by various enzymes:
  • Carbonic anhydrase: Nonenzymatic rate: $1.3 × 10^{-1} s^{-1}$, Enzymatic rate: $1 × 10^6 s^{-1}$, Rate enhancement: $7.7 × 10^6$
  • Chorismate mutase: Nonenzymatic rate: $2.6 × 10^{-5} s^{-1}$, Enzymatic rate: $50 s^{-1}$, Rate enhancement: $1.9 × 10^6$
  • Triose phosphate isomerase: Nonenzymatic rate: $4.3 × 10^{-6} s^{-1}$, Enzymatic rate: $4300 s^{-1}$, Rate enhancement: $1.0 × 10^9$
  • Carboxypeptidase A: Nonenzymatic rate: $3.0 × 10^{-9} s^{-1}$, Enzymatic rate: $578 s^{-1}$, Rate enhancement: $1.9 × 10^{11}$
  • AMP nucleosidase: Nonenzymatic rate: $1.0 × 10^{-11} s^{-1}$, Enzymatic rate: $60 s^{-1}$, Rate enhancement: $6.0 × 10^{12}$
  • Staphylococcal nuclease: Nonenzymatic rate: $1.7 × 10^{-13} s^{-1}$, Enzymatic rate: $95 s^{-1}$, Rate enhancement: $5.6 × 10^{14}$

Enzymes Are Classified by Reaction Type

• Table 9.2 outlines enzyme classification based on reaction type:

  • Oxidoreductases: Catalyze oxidation-reduction reactions.
  • Transferases: Facilitate the transfer of functional groups.
  • Hydrolases: Catalyze hydrolysis reactions.
  • Lyases: Catalyze group elimination to create double bonds.
  • Isomerases: Catalyze isomerization reactions.
  • Ligases: Catalyze bond formation coupled with ATP hydrolysis.
  • Translocases: Facilitate the movement of molecules across or within membranes.

• Enzymes are named by adding the suffix "-ase" to the substrate name or a phrase describing their action, such as urease for urea hydrolysis and alcohol dehydrogenase for alcohol oxidation.

• Systematic classification categorizes enzymes based on the chemical reactions they catalyze.

Enzymes Act on Specific Substrates

• Enzymes exhibit stereospecificity, such as aconitase in the citric acid cycle.
• Geometric specificity varies; some enzymes are absolutely specific to one compound, while others act on a range of related compounds with different efficiencies, like alcohol dehydrogenase.

Some Enzymes Require Cofactors

• Proteins' functional groups participate in acid-base reactions, form transient covalent bonds, and engage in charge-charge interactions but require cofactors for oxidation-reduction and group-transfer processes.
• Cofactors include metal ions like $Cu^{2+}$, $Fe^{3+}$, and $Zn^{2+}$, and organic molecules like $NAD^+$ and $NADP^+$.
• Permanently associated cofactors include biotin.
• A catalytically active enzyme-cofactor complex is called a holoenzyme; the holoenzyme without the cofactor is an apoenzyme.

9.2: Enzymes Work by Lowering Activation Energy

• Enzymes catalyze reactions by lowering the activation free energy, $\Delta G^{\ddagger}$, which is needed to reach the transition state.
• Binding energy stabilizes the substrate in the active site, offsetting a significant portion of the activation energy.
• Enzymes preferentially bind the transition state of the catalyzed reaction.
• Chemical reactions often involve multiple steps with intermediates, transition states, and activation energy barriers; the step with the highest activation energy is the rate-determining step.
• Catalysts provide a reaction pathway with a lower free energy transition state.

Enzymes work by Lowering Activation Energy (Transition State Diagram)

• The activation energy for a nonenzymatic reaction is $\Delta G^{\ddagger}N$ and for an enzyme-catalyzed reaction is $\Delta G^{\ddagger}E$.
• The reaction coordinate diagram illustrates the energy changes during a reaction, with dips representing the binding of substrate and product to the enzyme.

9.3: Catalytic Mechanisms

• Enzymes utilize metal ion cofactors or organic coenzymes, which can be reversibly bound cosubstrates or permanently associated prosthetic groups, often derived from vitamins.
• Enzymes employ catalytic mechanisms like general acid and base catalysis, covalent catalysis, and metal ion catalysis.
• The active site arrangement allows catalysis through proximity and orientation effects and electrostatic catalysis.

Reduction of $NAD^+$ to NADH

• Only the nicotinamide ring is affected by reduction, represented as hydride transfer.

Types of Catalytic Mechanisms

• Acid-base catalysis
• Covalent catalysis
• Metal ion catalysis
• Proximity and orientation effects
• Preferential binding of the transition state complex

Metal Ion Cofactors Act as Catalysts

• Nearly one-third of enzymes require metal ions like $Fe^{2+}$, $Fe^{3+}$, $Cu^{2+}$, $Mn^{2+}$, or $Co^{2+}$ for activity.
• $Mg^{2+}$ and $Zn^{2+}$ can be structural or catalytic.
• Metal ions participate by:
  • Binding substrates to orient them properly.
  • Mediating redox reactions.
  • Stabilizing or shielding negative charges.
• In carbonic anhydrase, $Zn^{2+}$ polarizes $H<em>2O$, forming $OH^−$ which attacks $CO</em>2$, facilitated by His 64.

Acid–Base Catalysis

• General acid catalysis involves proton transfer to lower the transition state's free energy.
• Asp, Glu, His, Cys, Tyr, and Lys can act as acid or base catalysts with pK's near the physiological pH range.
• Enzymes arrange catalytic groups around substrates for concerted acid-base catalysis.
• Enzyme activity is sensitive to pH, influencing side chain protonation.

Effects of pH on Enzyme Activity

• Observed pK's provide clues to essential amino acid residues.
• The pK of an acid-base group can vary based on its microenvironment.
• pH effects may indicate enzyme denaturation rather than residue protonation.
• Site-directed mutagenesis or comparisons of enzyme variants are more reliable for identifying crucial residues.

$RNase A$ Is an Acid–Base Catalyst

• Bovine pancreatic $RNase A$ hydrolyzes RNA in the small intestine.
  1. His 12 (general base) abstracts a proton from RNA 2'-OH, promoting nucleophilic attack on phosphorus. His 119 (general acid) promotes bond scission by protonating the leaving group.
  2. Water enters, hydrolyzing the 2',3'-cyclic intermediate. His 12 acts as a general acid, and His 119 acts as a general base.

Covalent Catalysis

• Covalent catalysis accelerates reaction rates through transient catalyst-substrate covalent bond formation, often involving nucleophilic attack.
• Coenzymes like thiamine pyrophosphate and pyridoxal phosphate act as covalent catalysts.

Catalysis Can Occur through Proximity and Orientation Effects

• Enzymes facilitate reactions by:
  1. Bringing substrates into contact with catalytic groups (~5x rate enhancement).
  2. Orienting substrates properly (up to ~100x rate enhancement).
  3. Stabilizing the transition state via charged groups (electrostatic effects).
  4. Freezing out translational and rotational motions (up to ~$10^7$ rate enhancement).

Enzymes Catalyze Reactions by Preferentially Binding the Transition State

• Enzymes bind transition states more strongly than substrates or products.
• Transition state analogs are potent enzyme inhibitors, like proline racemase inhibited by planar proline analogs.

Lysozyme

• Lysozyme degrades bacterial cell walls. Structural knowledge comes from X-ray studies with substrate analogs. Glu 35 and Asp 52 promote hydrolysis via acid-base and covalent catalysis.

Serine Proteases

• Serine proteases (including chymotrypsin, trypsin, and elastase) catalyze peptide bond hydrolysis with different specificities.
  • Chymotrypsin: bulky hydrophobic residues (Phe, Trp, Tyr)
  • Trypsin: positively charged residues (Arg, Lys)
  • Elastase: small neutral residues (Ala, Gly, Val)
• Diisopropylphosphofluoridate (DIPF) reacts with Ser 195 of chymotrypsin, identifying it as the active site Ser.
• Organophosphorus compounds like DIPF are toxic nerve gases that inactivate acetylcholinesterase.

Serine Proteases (cont.)

• Transition state preferential binding is responsible for the catalytic efficiency of serine proteases.
• DIPF is an effective inhibitor due to its tetrahedral phosphate group mimicking the transition state.