Lecture 17 Notes

Kinetics of the Reaction

• The progress of the reaction can be followed using a chromogenic substrate.
• N-Acetyl-L-phenylalanine p-nitrophenyl ester is a substrate analogue, chemically similar to the natural substrate.
• The reaction releases a colored product called p-Nitrophenolate.
• The reaction can be measured using a spectrophotometer.
• p-Nitrophenolate absorbs light at 400 nm.
• The rate of reaction can be measured by observing A400 (absorbance at 400 nm) over time using a spectrophotometer.

Burst Phase and Steady-State Phase

• Initially, in the first few milliseconds, the product is released much faster, showing a burst of activity.
• After a few seconds, the reaction occurs in a steady state and follows Michaelis-Menton kinetics.

Two-Step Reaction

• The reaction occurs in two steps:
  1. The enzyme reacts with the substrate to form a covalent intermediate (fast).
  2. Water breaks down the intermediate to regenerate the free enzyme (slow).
• Some product is released quickly during the burst phase.
• Once all the enzyme has been acylated, the reaction has to wait until the enzyme is regenerated to make more product, resulting in a slow phase.

Catalytic Triad

• Serine 195, histidine 57, and aspartate 102 form a catalytic triad.
• His 57 accepts a proton from Ser 195, acting as a base.
• Asp 102 helps orient His 57 correctly.
• The serine O- ion becomes a very strong nucleophile, highly reactive to electron-deficient atoms.

Oxyanion Hole

• An oxyanion hole stabilizes the anion intermediate.
• The tetrahedral intermediate contains an oxyanion (C-O- group).
• Hydrogen bonds to backbone N-H groups stabilize the negative charge.

Catalytic Strategies

• Most enzymes use one or more of these strategies for catalysis:
  • Covalent catalysis
  • General acid-base catalysis
  • Metal ion catalysis
  • Catalysis by proximity and orientation of two substrates

Carbonic Anhydrase

• Carbonic anhydrase hydrates $CO_2$ to carbonic acid.
• $k_{cat} = 10^6 s^{-1}$
• Each molecule can catalyze a million reactions per second.
• Carbonic anhydrase has a $Zn^{2+}$ cofactor.
• $Zn^{2+}$ is bound in the active site and binds a molecule of $H_2O$.
• $CO<em>2$ binds in the active site next to the $H</em>2O$ bound to $Zn^{2+}$.
• $Zn^{2+}$ acts to stabilize the deprotonated form of the water molecule.
• It reduces the $pK<em>a$ of $H</em>2O$ from 15.7 to 7.0 enabling it to more readily form $OH^-$.
• Therefore makes it a strong nucleophile.

Carbonic Anhydrase Mechanism

1. $Zn^{2+}$ facilitates the loss of $H^+$ by $H_2O$, forming $OH^-$.
2. $CO_2$ binds next to the activated $OH^-$ group.
3. $OH^-$ ion attacks $CO<em>2$, forming $HCO</em>3^-$.
4. $HCO<em>3^-$ is released, and a new $H</em>2O$ binds the $Zn^{2+}$.

Clinical Insights - Carbonic Anhydrase in Blood

• Carbon dioxide ($CO_2$) is produced from respiring tissues.
• $CO_2$ must be transported to the lungs to be exhaled.
• $CO_2$ is hydrated to carbonic acid in red blood cells.
• Carbonic acid is a weak acid and dissociates to form bicarbonate ions.
• The pH of blood must be carefully regulated.
• Blood needs a buffer to maintain the pH.
• The effective $pK_a$ of carbonic acid is 6.4.
• The action of carbonic anhydrase helps to buffer the blood.

Cofactors

• Many enzymes require cofactors to function.
• Cofactors are non-protein molecules required for enzyme/protein function.
• Two types of cofactors:
  • Coenzymes – organic molecules derived from vitamins
  • Metal ions, e.g., $Mg^{2+}$, $Zn^{2+}$, $Cu^+$
• The protein part, without the cofactor, is called an apoenzyme.
• The protein-cofactor complex together is called a holoenzyme.
• Example: Malate dehydrogenase
  • Malate dehydrogenase protein alone + NADH
  • Apoenzyme + Coenzyme = Holoenzyme

Summary

• Enzymes are specific catalysts.
• Many enzymes require cofactors: coenzymes or metal ions.
• Enzymes speed up reaction rates but do not affect the free energy change of the reaction.
• Free energy change can be calculated from the equilibrium constant.
• Enzymes stabilize the transition state, reducing the activation energy and increasing the rate of reactions.
• Enzymes bind their substrates in the active site.

Enzyme Mechanisms Summary

• Many different enzymes use 4 main strategies to catalyze reactions.
• Chymotrypsin is a serine protease that has been studied by various methods.
• A catalytic triad of Ser, His, and Asp is involved in the mechanism.
• The reaction proceeds via a tetrahedral intermediate, stabilized by an oxyanion hole.
• Substrate specificity is provided by the amino acids in the specificity pocket.
• Different proteases use different strategies to create a strong nucleophile for the attack of the peptide bond.
• Carbonic anhydrase is a very active enzyme and has an important role in blood.
• Carbonic anhydrase uses a metal ion to generate a highly reactive $OH^-$ molecule.