enzyme vedio -- hasika

[Audio] RIBONUCLEASE (RNase) Structure, Function & Mechanism of Action Postgraduate Biochemistry Seminar RNase A model enzyme RNA → fragments.

[Audio] What is Ribonuclease? Enzyme that catalyzes cleavage of RNA phosphodiester bonds Essential for RNA degradation, processing and turnover Participates in RNA quality control and regulation Found across bacteria, plants and animals Long RNA strand RNase RNA fragments.

[Audio] RNase A — The Model Enzyme Classical model for studying enzyme catalysis Compact, highly stable globular protein Cleaves single-stranded RNA efficiently Belongs to the RNase A superfamily Well-characterized structure and active site RNase A α-helices + β-sheets Active site → Model for studying chemical catalysis.

[Audio] Structure of RNase A Approximately 124 amino acid residues Compact globular α + β fold Positively charged RNA-binding active-site region Key catalytic residues: His12, Lys41 and His119 RNase A active site His12 General base/acid Lys41 Charge stabilization His119 Proton transfer RNA phosphate / 2′-OH positioned for catalysis.

[Audio] Mechanism of Action — Two-Step Catalysis Step 1 Transphosphorylation 2′,3′-cyclic phosphate Step 2 Hydrolysis RNA substrate 3′-phosphate product.

[Audio] . MECHANISM OF ACTION OF RNase A OVERVIEW Substrate Binding RNase A RNA Substrate Cleaves on the 3'-side of pyrimidine residues (C, U) 6-0 His 12 NH Active Site Lys 41 THE TWO-STEP CATALYTIC CYCLE STEP 1: TRANSPHOSPHORYLATION (FORMATION OF CYCLIC INTERMEDIATE) STEP 2: 1. Water Activation His 119 HO Asp 121 HYDROLYSIS (REGENERATION OF ENZYME) 1. Binding and Activation General Base General Acid His 12 (deprotonated) His 12 abstracts proton from 2'-OH General Base His 12 (deprotonated) His 119 (protonated) General Acid His 119 (protonated) 2. Nucleophilic Attack and Pentavalent Transition State His 119 attacks P Lys 41 stabilizes negative charge -o-P-O OH 2',3'-cyclic phosphodiester intermediate 3. Leaving Group Dissociation His 119 General Base His 119 (deprotonated) NH His 119 abstracts proton from H20 General Base His 119 (deprotonated) Water molecule General Acid His 12 (protonated) 2. Nucleophilic Attack and Pentavalent Transition State His 119 OH- attacks cyclic P tip Lys 41 stabilizes 3. Regeneration and Dissociation His 12 His 12 protonates 2-0 leaving group o d His 119 protonates 5'-0 leaving group s o OH 3-Product with 5'-OH negative charge o -o-P-o ase o -04—0 ases o OH OH 3'-Product with 3'-Phosphate 5'-OH OH 5'-Product with 5'-OH OVERALL REACTION H20 RNA 3'-Phosphate Product 5'-OH Product.

[Audio] Step 1 — Transphosphorylation 1. RNA binds the active site 2. His12 activates the 2′-OH 3. 2′-O attacks the adjacent phosphate 4. His119 facilitates proton transfer to the leaving group 5. 2′,3′-cyclic phosphate intermediate forms 2′-OH P 2′-O⁻ 2′,3′-cyclic phosphate.

[Audio] Step 2 — Hydrolysis 2′,3′-cyclic phosphate H₂O Hydrolysis 3′-phosphate Water is positioned in the active site His119 participates in water activation/proton transfer Cyclic phosphate is opened Final product is a 3′-phosphate-containing RNA fragment.

[Audio] Complete RNase A Catalytic Cycle RNA substrate Substrate binding His12 activates 2′-OH Transphosphorylation 2′,3′-cyclic phosphate Hydrolysis by H₂O 3′-phosphate + product release His12: proton transfer | Lys41: charge stabilization | His119: proton transfer.

[Audio] Conclusion & References RNases are essential for RNA metabolism. RNase A is a classic model of enzyme catalysis. His12, Lys41 and His119 are key active-site residues. Catalysis proceeds through transphosphorylation followed by hydrolysis. A 2′,3′-cyclic phosphate intermediate is formed before the 3′-phosphate product. Key pathway RNA → cyclic phosphate → 3′-phosphate References Lehninger Principles of Biochemistry Berg et al., Biochemistry Raines RT. Ribonuclease A. Peer-reviewed literature on RNase A catalysis THANK YOU.