lecture 4: serine and threonine proteases

what do chymotrypsin, trypsin and elastase all have in common?

• all serine proteases with a similar amino acid sequence

• binds to the side chain of amino acids in the specificity pocket

• recognises the main chain to confirm the polypeptide and help orientate it for cleavage

difference between chymotrypsin, trypsin and elastase

different specificities:

• chymotrypsin cuts after large aromatic residues

• trypsin cuts after positively charged amino acids

• elastase cuts after small hydrophobic amino acids

enzyme activation by proteolysis

• the enzymes start out as inactive zymogens

• proteolytic cleavage of the zymogens to activate them

• proteolytic activation is not reversible so it requires close control (e.g. use of inhibitor proteins)

chymotrypsinogen → chymotrypsin

• chymotrypsinogen → trypsin cleaves chymotrypsin between arg25 and ile16 (the 2 pieces remain attached via disulfide bonds)

• π-chymotrypsin → autolysis to clip out 2 specific dipeptides (ser14 and arg15, thr147 and asn148)

• α-chymotrypsin

formation of the oxyanion hole from chymotrypsinogen to chymotrypsin

on cleavage by trypsin, the new NH3 on the ile16 pairs up with the side chain on asp194, altering the conformation of the main chain between residues 193 and 195. this changes the position of ser195 side chain, forming the correct geometry for the catalytic triad and orients the main chain amides of residues 193 and 195 to form the oxyanion hole

blood clotting activation cascade

• intrinsic pathway → triggered when blood comes into contact with a damaged surface

• extrinsic pathway → triggered by external trauma that releases tissue factor into the blood

this leads to the activation of factor x, causing serine proteases to cleave the arg-gly bind in soluble protein fibrinogen

how is hemophilia passed down

inherited from the father

what are proteasomes

intracellular, multi-subunit, cylinder-shaped complexes with an interior cave containing proteolytically active sites that belong to the N-terminal of threonine hydrolases

threonine peptidase at the heart of the proteasome mechanism

why are ubiquitin-proteasome pathways useful

they play a role in cytoplasmic turnover

what did proteasome inhibitors do?

exhibit apoptosis in some tumour derived cell lines

why does lys33 have a reduced pKa in a threonine peptidase

due to its hydrophobic environment, which allows it to be deprotonated and acts as a base

dipeptide boronic acid (boetezomib) as a proteasome inhibitor

boron is an electron deficient atom, the inhibitor works by reacting with the active site nucleophile to create a covalent bond. this gives a tetrahedral shape that mimics the transition state that occurs during protein breakdown

what is bortezomib used as a treatment for

multiple myeloma

positive feedback of the blood clotting cascade

thrombin actually goes back and activates earlier factors to accelerate and finish the clot

negative feedback of the blood clotting cascade

antithrombrin and TFPI neutralises the proteases so the clot does not grow indefinitely