Serine Proteases (Lec 2&3) Flashcards
When hydrolysing peptides, what is the name of the bond that is cleaved and what 2 molecules is it between?
Draw the cleavage reaction and the 2 products
Scissile bond between C=O and N-H
Give 5 examples of Ser proteases.
Chymotrypsin Trypsin Elastase Subtilisin (bacteria) Complement C1
Give the name of the MAIN specificity pocket and state where it is located in relation to the peptide (upstream/downstream of scissile bond).
S1 specificity pocket upstream to scissile bond
For chymotrypsin, trypsin and elastase describe the side chains they recognise right before cleavage and give examples of these side chains.
Chy - large aromatic (Phe, Tyr, Trp) Try - +vely charged (Lys, Arg) Ela - small hydrophobic (Ala)
Give the catalytic triad present in chymotrypsin and draw out how we obtain the alkoxide ion in this triad
Ser 195 His 57 Asp 102
Describe how Ser and His were identified in the AS of chymotrypsin before the structure was solved
Draw out the reaction of PMSF with Ser
Ser - PMSF (phenyl methane sulphonyl fluoride) - PMSF bound to enzyme via O - inactive enzyme which highlighted the crucial Ser His - TPCK (tosyl phenylalanine chloromethyl ketone) - irreversibly binds to the His after inducing a nucleophilic attack by His when it reaches the active site
How many domains are there in chymotrypsin enzyme and what is their shape.
Draw the rough overall structure of this enzyme (including the location of the oxyanion hole etc)
2 domains - above & below AS fold into a B barrel made up of 6 antiparallel B strands
Describe the components of the oxyanion hole and what does the oxyanion hole stabilise?
Gly193 and Ser195 amide Hs point in towards the AS and work to stabilise the O- formed in the tetrahedral E-S intermediate
Is there high/low sequence identity between Trypsin and Chymotrypsin and Trypsin and elastase?
around 40% sequence identity between each pair spatial conservation of the catalytic triad is apparent - all organised in roughly same orientations/in same space
Give the specificity pockets of Chy, Try and Ela.
Chy - sides = Ala,Gly, base = Ser (different to catalytic triad Ser) Try - sides = G,G base = Asp Ela - sides = Val,Thr - make the pocket bulky in order for the peptide to fit into the AS
Can Ser proteases recognise the main chain and if so how do they do this?
Yes they recognise main chain elements H bond to N-H for eg forming anti-parallel B sheet between substrate and protein
Draw the schematic for the Ser protease active site - including the oxyanion hole and specificity pocket.
Why must enzymes such as chymotrypsin be closely controlled?
because they are proteases they could hydrolyse cellular components
How are Ser proteases, such as chymotrypsin, activated? Describe this mechanism and state the master regulator. Remember to describe the mechanism in terms of catalytic triad and oxyanion hole activation.
All proteases are synthesised as inactive precursor zymogens - requires proteolytic cleavage for activation Master regulator = enteropeptidase that cleaves Trypsinogen -> Trypsin Trypsin can then act on other zymogens Chymotrypsinogen > pi Chymotrypsin (catalysed by Trypsin) which is a di peptide pi chymotrypsin activates other pi Chymotrypsin molecules eventually 3 peptides made (all connected via disulphides) free NH3+ group on terminal of one of the peptides can then interact with the carboxyl group of Asp194 this causes conformational change of main chain and catalytic triad becomes orientated correctly also amide Hs of Gly193 and Ser195 form the oxyanion hole ENZYME = ACTIVE
Describe convergent evolution in terms of 2 enzymes
Chymotrypsin and Subtilisin (bacterial protease) v different sequences and structures however both have same catalytic triad
both converge on the same soln and carry out the proteolytic cleavage
Draw a schematic of the Ser protease active site - including specificity pocket etc
