Enzymatic Catalysis Notes

Enzymatic Catalysis

General Properties of Enzymes

• Enzymes differ from ordinary chemical catalysts in:
  • Reaction rate
  • Reaction conditions
  • Reaction specificity
  • Control
• The unique physical and chemical properties of the active site limit an enzyme’s activity to specific substrates and reactions.
• Some enzymes require metal ions or organic cofactors.

Ways to Increase the Rate of Chemical Reactions

• Increasing the temperature: Not practical for biological systems because many thrive only within narrow temperature ranges.
• Increasing the concentrations of the reacting substances: Space inside and outside the cell is crowded and limited, and many essential reactants are scarce.
• Adding a catalyst: A substance that participates in the reaction yet emerges at the end in its original form. Living systems use catalysts called enzymes to increase the rates of chemical reactions.

Enzymes vs Non-Biological Catalysts

• Most enzymes are proteins, but a few are made of RNA (ribozymes).
• Enzymes contain a specific fraction of the structure where reactions take place – active site.
• Most enzymes work at mild conditions, whereas many chemical catalysts require extremely high temperatures and pressures for optimal performance.
• Activity of many enzymes are regulated so that the organism can respond to changing conditions or follow genetically determined programs.

Enzymes Exhibit High Reaction Rates

Enzymes Classified by Reaction Type

• Many enzymes are named after the reaction they catalyze
• A more precise classification scheme systematically groups enzymes in a four-level hierarchy and assigns each enzyme a unique number. For example, chymotrypsin is known as EC 3.4.21.1

Enzymes Act on Specific Substrates

• Enzymes are highly specific for their substrates due to the precise interactions within the active site.

Stereospecific Enzyme-Substrate Interactions

• Enzymes can distinguish between stereoisomers of a substrate.

Some Enzymes Catalyze Highly Stereospecific Reactions

• Aconitase catalyzes the conversion of citrate to isocitrate in a stereospecific manner.

Some Enzymes are More Permissive

• Chymotrypsin can catalyze the hydrolysis of peptide bonds and ester bonds.
  • $RC—NHR' + H<em>2O \rightarrow RC -0- + H</em>2NR'$
  • $RC OR' + H_2O \rightarrow RC 0+ HOR'$

Cofactors Expand the Range of Enzymatic Reactions

• Certain functional groups in the enzyme’s active site perform the same catalytic function as small chemical catalysts.
• In some cases, the amino acid side chains of an enzyme cannot provide the required catalytic groups, so a tightly bound cofactor participates in catalysis.
• Some enzyme cofactors are organic molecules known as coenzymes, which may be derived from vitamins.

Inorganic Ions as Cofactors

Coenzymes as Transient Carriers of Atoms or Functional Groups

Cofactors in Oxidation-Reduction Reactions

• Example of ethanol oxidation to acetaldehyde by alcohol dehydrogenase (ADH) using NAD+ as a cofactor:
  • $CH<em>3CH</em>2OH + NAD^+ \rightleftharpoons CH_3CH + NADH + H^+$
  • Ethanol is converted to acetaldehyde with the help of NAD+.

Structure & Reaction of NAD(P) Plus

• Nicotinamide adenine dinucleotide (NAD+)
• Nicotinamide adenine dinucleotide phosphate (NADP+)

Activation Energy and the Reaction Coordinate

• An enzyme provides a lower-energy pathway from substrate to product but does not affect the overall free energy change for the reaction.

The Activation Energy ($\Delta G$)

• In a biochemical reaction, the reacting species must come together, overcome the repulsion, and undergo electronic rearrangements that result in the formation of products.

Transition State

• The energy-requiring step of the reaction is shown as an energy barrier, called the free energy of activation or activation energy and symbolized $\Delta G^{\ddagger}$.
• The point of highest energy is known as the transition state. The height of the activation energy barrier determines the rate of a reaction (the amount of product formed per unit time).
• The higher the activation energy barrier, the less likely the reaction is to occur (the slower it is). Although the reactant molecules have varying free energies, very few of them have enough free energy to reach the transition state during a given time interval.
• The lower the energy barrier, the more likely the reaction is to occur (the faster it is), because more reactant molecules happen to have enough free energy to achieve the transition state during the same time interval.
• Thermodynamic calculations indicate that lowering $\Delta G^{\ddagger}$ by about 5.7 kJ · mol–1 accelerates the reaction 10-fold. A rate increase of $10^6$ requires lowering $\Delta G^{\ddagger}$ by six times this amount, or about 34 kJ · mol–1

Transition State Diagram

• Illustrates the energy changes during a reaction.
• Includes the activation energy $\Delta G^{\ddagger}$ and the free energy change of the reaction $\Delta G_{reaction}$.

Effect of a Catalyst

• A catalyst lowers the activation energy $\Delta G^{\ddagger}$ but does not change the overall free energy change of the reaction $\Delta G_{reaction}$.
• The reduction in $\Delta G^{\ddagger}$ by the catalyst is denoted as $\Delta \Delta G^{\ddagger}$.

Catalytic Mechanisms

• Amino acid side chains that can donate or accept protons can participate in chemical reactions as acid or base catalysts.
• Nucleophilic groups can catalyze reactions through the transient formation of covalent bonds with the substrate.
• In metal ion catalysis, the unique electronic properties of the metal ion facilitate the reaction.
• Enzymes accelerate reactions by bringing reacting groups together and orienting them for reaction.
• Transition state stabilization can significantly lower the activation energy for a reaction.

Three Kinds of Chemical Catalytic Mechanisms Used by Enzymes

• Acid-base catalysis: A proton is transferred between the enzyme and the substrate.
  • Divided into acid catalysis and base catalysis.
• Covalent catalysis: A covalent bond forms between the catalyst and the substrate during formation of the transition state.
• Metal ion catalysis: Metal ions mediate oxidation–reduction reactions or promote the reactivity of other groups in the enzyme’s active site through electrostatic effects.

Mechanisms of Keto-Enol Tautomerization

• Involves the interconversion of a ketone (keto form) and an enol (alcohol form with a double bond).

Effects of pH on Enzyme Activity

• Enzyme activity is often pH-dependent.

The RNase A Mechanism

• RNase A uses acid-base catalysis to hydrolyze RNA.
• His 12 acts as a general base, and His 119 acts as a general acid.

Schiff Base Formation

• Involves the reaction between an amine and a carbonyl compound to form an imine.

Decarboxylation of Acetoacetate

• Illustrates the role of Schiff base formation in decarboxylation reactions.
  • $CH<em>3-C(=O)-CH</em>2-COO^- \rightarrow CO<em>2 + CH</em>3-C(=O)-CH_3$

Biologically Important Nucleophilic Groups

• Examples include hydroxyl, sulfhydryl, amino, and imidazole groups.

Biologically Important Electrophilic Groups

• Examples include protons, metal ions, carbonyl carbon atoms, and cationic imines (Schiff base).

Role of $Zn^{2+}$ in Carbonic Anhydrase

• $Zn^{2+}$ facilitates the reaction between $CO<em>2$ and $H</em>2O$.

Catalysis via Proximity & Orientation

• Enzymes bring reactants together and orient them appropriately for the reaction.

Geometry of an $S_N2$ Reaction

• Enzymes can facilitate $S_N2$ reactions by positioning reactants for backside attack.

Catalysis via Preferential Transition State Binding

• Enzymes preferentially bind the transition state, lowering the activation energy.

Inhibition by Transition State Analogs

• Transition state analogs are compounds that resemble the transition state of a reaction and can act as potent inhibitors.

Serine Proteases

• The catalytically active Ser, His, and Asp residues of serine proteases were identified by chemical labeling and structural analysis.
• A binding pocket determines the substrate specificity of the various serine proteases.
• Serine proteases catalyze peptide bond hydrolysis via proximity and orientation effects, acid–base catalysis, covalent catalysis, electrostatic catalysis, and transition state stabilization.
• Zymogens are the inactive precursors of enzymes.

The Catalytic Triad of Chymotrypsin

• The hydrogen-bonded arrangement of the Asp, His, and Ser residues of chymotrypsin and other serine proteases is called the catalytic triad.
• Asp 102 promotes catalysis by stabilizing the resulting positively charged imidazole group of His 57.

DIPF Irreversibly Inactivates Serine Proteases

• Diisopropylphosphofluoridate (DIPF) reacts with the active site serine residue, leading to inactivation.

Affinity Labeling

• Technique used to identify active site residues.
• Examples: Tosyl-L-lysine chloromethylketone for trypsin and Tosyl-L-phenylalanine chloromethylketone (TPCK) for chymotrypsin.

Specificity Pockets of Serine Proteases

• Different serine proteases have different specificity pockets, allowing them to bind and cleave different substrates.

Divergent Evolution

• Chymotrypsin, trypsin, and elastase have similar structures due to divergent evolution.

Convergent Evolution

• Subtilisin is an example of convergent evolution, with a similar catalytic triad but no sequence similarity to chymotrypsin.

Mechanism of Serine Proteases

1. General base catalysis and nucleophilic attack to form a tetrahedral intermediate.
2. General acid catalysis aids breakdown of the tetrahedral intermediate to the acyl-enzyme intermediate.
3. Amine product is released and replaced by water.
4. General base catalysis and nucleophilic attack to form a tetrahedral intermediate.
5. General acid catalysis aids breakdown of the tetrahedral intermediate to the carboxyl product and the active enzyme.

TS Stabilization in Serine Proteases

• The oxyanion hole stabilizes the tetrahedral intermediate.

Protease Inhibitors Limit Protease Activity

• Examples include pancreatic trypsin inhibitor (BPTI).

Elastase: Acyl-Enzyme & Tetrahedral Intermediates

• Illustrates the structures of the acyl-enzyme and tetrahedral intermediates.

Zymogens

• Proteases are synthesized as inactive precursors (zymogens).
• Activated by proteolysis.
• Example: Activation of trypsinogen to trypsin.
  • Trypsinogen + enteropeptidase or trypsin --> Val-(Asp)4— Lys + Trypsin

Blood Coagulation Cascade

• Involves a series of zymogen activations.