Enzyme Catalytic Mechanisms

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Presentation transcript:

Enzyme Catalytic Mechanisms Dr. Kevin Ahern

Chymotrypsin Serine Proteases Cleave Peptide Bonds Specificity of Cutting Common Active Site Composition/Structure Mechanistically Well Studied Chymotrypsin

Chymotrypsin Catalysis H2O

Chymotrypsin Yellow Color p-nitrophenolate N-Acetyl-L-phenylalanine p-nitrophenyl ester

Serine Proteases

Serine Proteases Folded Polypeptide Chain of Enzyme Catalytic Mechanism Catalytic Triad of Active Site Substrate for Enzyme

Determines What Substrate Serine Proteases Catalytic Mechanism Region of Enzyme That Determines What Substrate the Enzyme Binds

Serine Proteases 2. Structural Changes Induced by Binding Catalytic Mechanism 2. Structural Changes Induced by Binding Change Electronic Environment of Catalytic Triad 1. Binding of Substrate Stimulates Slight Structural Changes

N abstracts Proton From Serine Proteases Catalytic Mechanism N abstracts Proton From Serine’s Side Chain, Creating Alkoxide Ion

Serine Proteases Alkoxide Ion Makes Nucleophilic Attack Catalytic Mechanism Alkoxide Ion Makes Nucleophilic Attack on Carbonyl Carbon of Peptide Bond

Serine Proteases Peptide Bond Broken as N Binds to H on Histidine Catalytic Mechanism Tetrahedral Intermediate Stabilized by Oxyanion Hole

Serine Proteases Fast Phase of Catalysis Completed Half of Polypeptide Released from Enzyme Catalytic Mechanism Other Half of Polypeptide Covalently Linked to Serine Fast Phase of Catalysis Completed

Serine Proteases 2. Nitrogen Attacks Proton of Water Catalytic Mechanism 2. Nitrogen Attacks Proton of Water 3. Hydroxide Attacks Carbonyl Carbon 1. Water Enters Active Site

Serine Proteases Oxygen Abstracts H on N-Group, Breaking Bond with Serine Catalytic Mechanism Oxyanion Hole Stabilizes Tetrahedral Intermediate

Slow Phase of Catalysis Complete Serine Proteases Catalytic Mechanism Slow Phase of Catalysis Complete

Serine Proteases Cycle Complete. Enzyme Ready to Start Anew Enzyme Returned to Original State Catalytic Mechanism Other Polypeptide Fragment Released Cycle Complete. Enzyme Ready to Start Anew

Serine Proteases 1. Binding of substrate to S1 pocket Catalytic Mechanism 1. Binding of substrate to S1 pocket 2. Formation of alkoxide ion 3. Nucleophilic attack Stabilization of intermediate 4. Breakage of peptide bond 5. Release of peptide 1 6. Entry and activation of water 7. Release of peptide 2 from enzyme

S1 Pockets and Specificities Phenylalanine Tryptophan Tyrosine Glycine Alanine Valine Lysine Arginine

Serine Proteases Site-directed mutagenesis No mutation - activity = 100 Serine to alanine - activity = 0.00001 Histidine to alanine - activity = 0.00001 Aspartic acid to alanine - activity = 0.001 All three to alanine - activity = 0.00001 No enzyme - activity = 0.000000001

Protein Cleavage Agents Subtilisin - C-terminal side of large uncharged side chains Chymotrypsin - C terminal side of aromatics (Phe, Tyr, Trp) Trypsin - C-terminal side of lysine and arginines (not next to proline) Carboxypeptidase - N-terminal side of C-terminal amino acid Elastase - Hydrolyzes C-side of small AAs (Gly, Ala) Cyanogen Bromide (chemical) - Hydrolyzes C-side of Met

Inhibiting Protease Action Serpins = Serine Protease Inhibitors α-1-antitrypsin

Reactive oxygen species

Prevalence of α-1-antitrypsin deficiency α-1-antitrypsin deficiency by %

The New Serine Protease Song (to the tune of “Rudolph the Red-Nosed Reindeer”) Copyright © Kevin Ahern Metabolic Melody (Intro) All serine proteases Work almost identically Using amino acid Triads catalytically   First they bind peptide substrates Holding onto them so tight Changing their structure when they Get them in the S1 site Then there are electron shifts At the active site Serine gives up its proton As the RE-ac-tion goes on Next the alkoxide ion Being so electron rich Grabs peptide’s carbonyl group Breaks its bond without a hitch   So one piece is bound to it The other gets set free Water has to act next to Let the final fragment loose Then it’s back where it started Waiting for a peptide chain That it can bind itself to Go and start all o’er again