Lecture 19 Flashcards
Enzyme substrate complex
- substrate bind at the active site of the enzyme
- conformational changes in the substrate aided by bonding interactions in the active site result in the formation of the transition state
- products are released after bond cleavage
What does “electron pushing” mean in a reaction mechanism
flow of electrons from one atom to another
How do electrophiles and nucleophiles behave in a chemical reaction
nucleophiles attack electrophiles by donating electron pairs to the electrophilic center
Explain base-catalysis in the context of the chymotrypsin-catalyzed reaction
- catalytic histidines can abstract a proton away from catalytic serines and also from water, activating their oxygens to act as nucleophiles
Explain covalent catalysis in the context of the chymotrypsin-catalyzed reaction
- protein groups serve as covalent catalysts due to the presence of lone electron pairs
- in active sites of some enzymes these groups become deprotonated and act as nucleophiles
- attack electron-deficient atoms on substrates and form covalent intermediates
- chymotrypsin use their activated serine alkoxide ion to perform a nucleophilic attack on the peptide bond, forming a covalent intermediate as a first step in hydrolyzing that bond
What are serine proteases, cleave how? where?
scissile bond? active site? which residues are important?
- enzymes that cleave peptide bonds in proteins
- cleave by cutting/hydrolyze peptide bonds
- cleave at C terminal to bulky, hydrophobic side chain
- scissile, peptide bond that is cleaved
- active site, site in enzyme where catalysis occurs
- important residues, Ser195, His57, and Asp102 as well as the residues in the hydrophobic pocket and in the oxyanion hole
What is the overall structure and role of chymotrypsin
- globular protein with an active site containing a catalytic triad (His57, Asp102, Ser195)
- a hydrophobic specificity pocket and an oxyanion hold
- chymotrypsin binds bulky hydrophobic side chains via its hydrophobic specificity pocket
What is the role of the catalytic triad
- His57 acts as a “base catalyst” abstracting a proton from Ser195-OH
- the protonated positively charged His57 is now stabilized by Asp102
- Ser195-O- now acts as a “covalent catalyst” - forms a covalent bond with the substrate while hydrolyzing the peptide bond
How does hydrolysis of peptide bonds by chymotrypsin take place
- the enzyme binds to the protein substrate (ES); the enzyme forms a “covalent intermediate” with the substrate, hydrolyzing the peptide bond in the process; one of the products, P1, is released - fast step- the remainder of the substrate, P2, remains bound temporarily in the enzyme active site (E-P2)
- The second product, P2, is released from the enzyme - slow step
Explain how the catalytic triad activates Ser195 for nucleophilic attack of the carbonyl carbon of the scissile peptide bond
- serine is particularly reactive because of its position within the catalytic triad
- the interaction between Ser195, His57, and Asp102 in the active site of the chymotrypsin activates the serine, forming an alkoxide ion (OH -> O-), a very strong nucleophile
What are the products of peptide bond cleavage, the order of release of each product from the enzyme active site
- first product 1 is released (carboxyl terminal fragment), and then product 2 (amino terminal fragment)
Explain the formation of the tetrahedral intermediate following nucleophilic attack of the carbonyl carbon, first by the Ser195 alkoxide ion, and second by water
- The formation of the tetrahedral intermediate begins with the nucleophilic attack of the Ser195 alkoxide ion on the carbonyl carbon of the peptide bond, forming an acyl-enzyme intermediate
- a water molecule attacks the carbonyl carbon, facilitated by a base (often His57), leading to cleavage of the peptide bond and release of the C-terminal fragment from the enzyme active site.
Explain how the negatively charged oxyanion is transiently stabilized by the oxyanion hole in the enzyme active site
oxyanion is stabilized in oxyanion hole made by backbone amino groups of Gly193 and Ser195
Explain the sequence of events in chymotrypsin-catalyzed reaction mechanism step-by-step
- polypeptide substrate binds to enzyme active site
- His57 removes a proton from Ser195, which allows a nucleophilic attack by the serine oxygen on the carbonyl carbon of the peptide
- His57 donates a proton to the amino group of the substrate, resulting in peptide bond cleavage. The carboxyl-terminal fragment is released as the first product
- Water enters the active site. His57 acts as a general base and removes a proton from water. The resulting OH- acts as a nucleophile and attacks the carbonyl carbon of the covalent acyl-enzyme intermediate
- His57 donates a proton to Ser195, resulting in cleavage of the acyl-enzyme intermediate. The amino-terminal fragment is released as the second product, and the catalytic triad is regenerated
- The functional catalytic triad is regenerated within the enzyme active site
oxyanion
- stabilized in oxyanion hole
