Serine Proteases Flashcards
What bonds do serine proteases cleave?
Peptide bonds.
What is the catalytic triad?
A combination of ser, asp and his residues found in the active site of all serine proteases. His acts as a general base to remove a proton from Ser, making it a better nucleophile. Asp stabilises His by electrostatic interaction.
How was Ser-195 in the catalytic triad discovered?
By using DIPF as a suicide substrate.
How was His-57 in the catalytic triad discovered?
By affinity labelling, using TPCK and chymotrypsin.
How does the catalytic triad show convergent evolution?
It has been discovered in many enzymes, with the amino acids in slightly different positions, depending on the substrate of that enzyme.
What is the oxyanion hole?
It preferentially binds the transition state in serine proteases.
What are some similarities between the catalytic mechanisms of serine proteases and other proteases (e.g. Asp proteases)?
Hydrolysis of the peptide bond is favoured by:
- presence of a nucleophile to attack the carbonyl group
- presence of charges to polarise the carbonyl group and to stabilise the tetrahedral intermediates
- presence of a proton donor to make the amine group a better leaving group.
What is the scissile bond?
The specific bond cleaved by the enzyme.
What is the S1 site?
The binding site on the serine protease for the P1 amino acid side chain.
What is the S1 pocket of chymotrypsin like?
It doesn’t contain any specific side chains and can bind bulky amino acids in the P1 position, such as phe/trp/Tyr.
What is the S1 pocket in trypsin like?
S1 pocket contains an Asp residue, and binds positively charged amino acids in the P1 position- Arg/Lys
What is the S1 pocket in elastase like?
It is an occluded pocket with restricted access, due to having two Val side chains on either side of the pocket. This means that is can bind small/neutral amino acids in the P1 position- Ala/Gly/Ser/Val
How was the chymotrypsin mechanism studied?
By pre-steady state kinetics. Used an artificial substrate with a Phe residue in the P1 position and an acetyl group attached to mimic the peptide backbone. An ester was used as cleavage is slower and easier to follow. When the artificial cleavage occurs, phenolate is formed which can be followed by its yellow colour.
What did the chymotrypsin mechanism experiments show?
Showed biphasic kinetics.
1) initial burst phase where the product is formed in equal amounts to the enzyme.
2) steady state phase where the product is produced at a constant rate dependent on enzyme concentration.
This is an example of ping-pong kinetics.
What is a zymogen?
Large inactive precursor molecules of serine proteases which are activated by proteolytic cleavage in the gut.
How is trypsinogen activated?
Activated by cleavage after lys15, catalysed by enteropeptidase which is under hormone control. The trypsin formed can then cleave more trypsinogen.
How is unwanted trypsinogen activation prevented in the pancreas?
By a pancreatic trypsin inhibitor which acts as a transition state analogue and binds tightly to trypsin.
How is proelastase activated?
Via a similar mechanism to trypsinogen activation.
How is chymotrypsinogen activated?
Activated by trypsin cleavage. Partially active pi-chymotrypsin is formed after trypsin cleaves after an Arg residue. Fully active alpha-chymotrypsin is formed after chymotrypsin cleavage causes the removal of two dipeptides from the molecule.
How do serine proteases show divergent evolution?
Trypsin, chymotrypsin and elastase are all very similar but have different cleavage specificities.
Where are zymogens secreted from?
The acinar cells of the pancreas.
What type of enzyme control is zymogen synthesis and cleavage an example of?
Covalent modification.
