Lecture 3.2: Enzyme Reaction Mechanisms Flashcards
Features of Catalytic Reactions Mechanisms
- show the optimized microenvironment: substrates bind to active site through noncovalent weak interactions
- illustrate the principle of transition state stabilization
- Give the classic examples of acid-base & covalent catalysis
- informed design of HIV protease and SARs coronavirus protease inhibitors
Serine protease mechanisms involves [ ] to cleave peptide bonds
acid-base catalysis
covalent catalysis
Serine protease family includes
chymotrypsin
trypsin
elastase
conserved tertiary structure despite limited sequence similarity
The active sites of serine proteases share common structural components: What are they?
binding pocket
catalytic triad
oxyanion hole
Binding pocket
determine substrate specificity
catalytic triad
3 conserved amino acid residues
Oxyanion hole
active site close to catalytic triad
negatively charged O2 [transition state during rxn]
Pocket Properties?
Chymotrypsin
deep, hydrophobic
made of 2 glycines + serine
Pocket properties?
Trypsin
deep pocket, negative charge at bottom
made of 2 glycines + 1 aspartate
pocket properties
Elastase
shallow, hydrophobic pocket
made up of 1 threonine + 1 valine + 1 serine
smaller than other 2
What amino acids bind to chymotrypsin?
phenylalanine
tyrosine
tyrptophan
aromatic + bulky
What amino acids bind to trypsin?
lysine
arginine
NOT histidine because too bulky
what amino acids bind to elastase?
glycine
alanine
All serine proteases use catalytic triad consisting of three [ ] amino acids: [ ]
conserved
ser, his, asp
What two amino acids function together to make Ser into a highly reactive nucleophile?
Asp and His
Asp and His Steps to making Ser a highly reactive nucleophile
- Asp makes His imidazole side chain more basic (a negative charged oxygen stabilizes His and makes more basic His residue)
- His removes proton from Ser making Ser more nucleophile (His acts as good base catalyst and removes proton from ser anc creates negative charge oxygen)
- Serine can then act as nucleophile (break covalent peptide bond)
The tetrahedral intermediate is stabilized by [ ] with backbone [ ] groups in the [ ]
hydrogen bonds
amide
oxyanion hole
Chymotrypsin Reaction Mechanism:
Step 1
- substrate binds to the active site after an interaction between the aromatic side chain and specificity pocket
- polypeptide substrate binds to enzyme active site
- substrate binds to enzyme with high specificity
Chymotrypsin Reaction Mechanism:
Step 2
- nucleophilic attack by the Ser oxygen on the carbonyl carbon results in the formation of an acyl-enzyme intermediate
- His57 removes a proton from Ser 195 –> nucleophillic attack by Serine
- Oxyanion is stablized in oxyanion hole made by backbone amino acid groups gly193 and ser195
Chymotrypsin Reaction Mechanism:
Step 3
- His57 donates a proton to the amino group of substrate, facilitating peptide bond cleavage
- His 57 donates a proton to the amino group of the substrate, resulting in peptide bond cleavage
- product 1 released
Chymotrypsin Reaction Mechanism:
Step 4
- the nucleophilic attack by OH- on the carbonyl carbon leads to the formation of 2nd tetrahedral intermediate
- water enters the active site
- His57 = general base
- oxyanion hole stabilized
Chymotrypsin Reaction Mechanism:
Step 5 & 6
- His57 donates a proton to Ser195 resulting in cleavage of acyl-enzyme intermediate and regeneration of the catalytic triad
- Amino-terminal fragment is released as the second product
Key Points of Chymotrypsin Reaction Mechanism:
positioning of catalytic triad amino acids in the active site creates the [ ] that can cleave the peptide bond
nucleophile
Key Points of Chymotrypsin Reaction Mechanism:
positioning of Gly193 and Ser195 form a [ ] which stabilizes [ ]
microenvironent (oxyanion hole)
an otherwise unstable tetrahedral intermediate
Key points of Chymotrypsin Reaction Mechanism:
His serves as [ ] catalytst where it withdraws a proton to increase the [ ] of [ ] and donates a proton to resolve the [ ] tetrahedral intermediate
general acid-base
nucleophilicity of Ser
2nd tetrahedral intermediate
HIV Protease
Uses which two types of catalysis
acid-base
covalent catalysis
Enolase Reaction Mechanisms
Uses which two types of catalysis to do what?
general acid-base catalysis and metal ion catalysis to eliminate a water molecule
Mg2+ Metal Ion Catalysis
Mg2+ ions orient the [ ] in the [ ] and stabilized the [ ] intermediate
substrate
active site
carbanion
