Enzyme mechanisms: Chymotrypsin, Lysozyme and HIV protease Flashcards
Protease
Proteases catalyze the hydrolysis of polypeptide backbones to yield smaller peptides
What/where does trypsin cleave?
cleaves C terminal side of R,K
What/where does chymotrypsin cleave?
cleaves C terminal side of F,Y,W
What/where does elastase cleave?
cleaves smaller, non-polar amino acids A,G,S,V
What is the catalytic triad of chymotrypsin?
Ser195 and His57 plus a third invariant residue in serine proteases is Asp102, which is buried in a solvent-inaccessible pocket.
What is the first step in the serine protease mechanism?
- Binding and formation of ES complex
What gives serine protease specificity to its substrate?
There is a hydrophobic pocket in the active site that conforms to the aromatic ring in the peptide to be cleaved
What is the second step in the serine protease mechanism?
- Nucleophilic attack by Ser OH.
A. His57 deprotonates Ser195 OH – general base catalysis
B. Ser O- attacks carbonyl carbon (nucleophilic) – proximity and orientation
C. Forming tetrahedral intermediate 1 – covalent catalysis
What is the third step in the serine proetase mechanism?
- Protonation of peptide amino group.
His 57 protonates peptide amino group – general acid catalysis
Peptide bond is cleaved
N-terminal fragment still attached to Ser 195 (acyl- enzyme intermediate)
Neutron diffraction shows that the proton between His57 and Asp102 remains with His (electrostatic catalysis)
What is the fourth step in the serine protease mechanism?
- Deprotonation of H2O and attack carbonyl carbon (H2O is second substrate)
A. His57 acts as a base to abstract proton from H2O – general base catalysis.
(note: pH must be high enough so that His57 isn’t protonated!)
B. Resulting hydroxyl ion attacks carbonyl forming tetrahedral intermediate 2.
What effect does pH have on the serine protease mechanism?
pH must be high enough so that His57 isn’t protonated!
Step 4 of the mechanism requires His57 to be deprotonated so that it can abstract a proton from the aqueous solute so that hydroxyl ion can attack the substrate and form tetrahedral intermediate 2.
Which intermediate in the serine protease mechanism is the least stable?
Intermediate 2
It is stabilised by the oxianion hole which accomidates the oxyanion in tetrahedral intermediate 2
Peptide N’s form good hydrogen bonds with oxyanion, but not when carbonyl is in a trigonal conformation, e.g. ES complex or acyl-enzyme intermediate
Thus the oxyanion hole enables preferential binding to the transition state
oxyanion hole in serine protease
The oxianion hole accomidates the oxyanion in tetrahedral intermediate 2 and stabilses it
Peptide N’s form good hydrogen bonds with oxyanion, but not when carbonyl is in a trigonal conformation, e.g. ES complex or acyl-enzyme intermediate
Thus the oxyanion hole enables preferential binding to the transition state
Explain the catalytic rate of serine protease in relation to pH
chymotrypsin His57 pKa ~ 7
