lecture recording on 13 March 2025 at 17.03.59 PM

Mechanism Understanding

• It is important to understand the mechanisms behind chemical reactions rather than just memorizing them.

• Homework assignments will feature mechanism questions similar to exam questions, providing the opportunity to practice.

• Students are encouraged to work through blank notes and understand each step of the mechanisms presented.

RNase A Enzyme

• Function of RNase A: Cleaves RNA molecules, breaking phosphodiester bonds.

• Active Site: Illustrates important residues for catalysis—specifically, histidine residues.

• Substrate: RNA molecule, often represented in parts due to its lengthy structure.

Step 1: Proton Transfer

• In the active site, a histidine residue acts as a catalytic base and deprotonates the 2′-hydroxyl group of ribose.

• This results in the formation of a negative charge on oxygen (O−), which is a strong nucleophile.

Step 2: Nucleophilic Attack

• The O− will seek out an electrophile, specifically the phosphorus in the phosphate group.

• Transition from protonated to deprotonated states is crucial for reactivity.

Step 3: Bond Rearrangements

• As O− attacks phosphorus, existing bonds may shift, leading to the formation of a transition state.

• At this stage, bonds are broken, and charge distributions stabilize through interactions with nearby residues (e.g., arginine).

Transition State Stabilization

• Important because transition states are fleeting and not considered intermediates.

• Stabilization can involve ionic interactions with charged groups in the active site.

Phosphodiester Bond Cleavage

• Dismantling the bonds presents a mechanism that allows RNA to be cleaved efficiently.

• The breakdown of the transitional state results in the departure of products, including an actual substrate segment.

Role of Histidines

• One histidine acts as a base (deprotonating), while another acts as an acid (protonating) during the reaction process.

• This dual role is vital for facilitating the cleavage and ensuring the reaction proceeds smoothly.

Specificity in Enzyme Mechanisms

• Catalytic Triad: Many proteases share this concept, indicating that they utilize similar mechanism basics but differ in substrate specificity.

Example Proteases

• Chymotrypsin: Cleaves after bulky aromatic residues (e.g., phenylalanine, tyrosine).

  • Its specificity pocket is adapted for accommodating larger side chains.

• Trypsin: Targets lysine and arginine, featuring charged residues to accommodate these basic amino acids.

• Elastase: Focuses on smaller aliphatic residues like alanine and valine.

Insights on Catalysis

• All reviewed enzymes, while employing common mechanisms, have different active site compositions that dictate specificity.

• General principles include proton transfers, charge stabilizations, and bond shifts sit at the heart of enzymatic actions.

Kinetics Introduction

• Enzyme kinetics measures how quickly reactions take place, typically represented in product formation versus time graphs.

• Vmax: maximum velocity when all active sites are filled.

• Km: substrate concentration at half-maximal velocity, indicating affinity.

Kcat

• Defined as the turnover number per active site per second, derived from Vmax and enzyme concentration. Higher Kcat values denoting more efficient catalysis.

Catalytic Efficiency

• Evaluated as the ratio of Kcat to Km, reflecting how effectively an enzyme converts substrate to product. High values indicate better enzyme performance.

Michaelis-Menten Model

• Describes enzyme kinetics by forming enzyme-substrate (ES) complexes and categorizing their conversions to products based on discrete rates (k1, k-1, and k2).

• Breakdown of ES complexes helps quantify both binding affinity and reaction rates.