7: SERINE PROTEASES Flashcards
Designing a protease
- protease attacks and cleaves peptide bond using water (hydrolase)
- hydrolysis reaction to make carboxylate and free amino
- spontaneous process ^G is -ve but its under kinetic control so ^G for uncatalysed reaction is v.high
designing a protease mechanism
- substitution reaction; replace NH2 with OH
- C starts sp2 trig.planar - sp3 tetra. intermediate - sp2 again
- Nu attack on C forms oxyanion intermediate; decomposition to reform carbonyl group and L is ejected as L-
increase reactivity of protease mechanism
good nucleophile:
-high pKa species; OH»_space; H2O; deprotonated; naked charge
good leaving group:
-low pKa; neutralise/protonate so becomes LH
-serine side chain acts as nucleophile; to improve reactivity of ser OH, have to have a base to pull of proton to generate charge on oxygen so it becomes a better nucleophile
catalytic triad
- Ser195 nucleophile + His57 Base + deprotonated Asp102
- usually Ser side chain has v.high pKa so its hard to pull proton off because resulting conj. base (O) wants proton back
- needs a base to deprotonate Ser to make better nucleophile
- His57 acts as B to pull proton off; but usual pKa of His is 6/7 which is too low as a base to pull proton off
- needs to be activated to make it a better base; role of Asp102; in deprotonated form can form v.good charge-charge interactions through H-bond to stabilise protonated His side chain
on catalytic triad
- refers to position of residues in primary structure of chymotrypsin
- when the enzyme folds up, they come together in space to form the active site
identification of catalytic triad
serine 195:
- chemical modification experiments with DIPF
- Ser Nu attack on P and eject fluorine as F- to form a covalent aduc
- irreversibly inhibited, so can investigate which ser side chain has been modified
his57:
- affinity labelling using a substrate analog TPCK
- TPCK resembles substrate for chymotrypsin but is chemically modified so that it reacts irreversibly with the enzyme when bound in the active site
detailed mechanism of serine protease
- S comes into active site
- Nu attack by Ser on C=O to form oxyanion sp3 tetragedral intermediate
- to do this, His acts as B; Asp exerts electrostatic effect
- decomposition to give acyl-enzyme intermediate
- His acts as acid to form NH2 leaving group; as it leaves, water comes into active site to hydrolyse ester linkage
- His acts as B to promote Nu attack by water to form second tetra intermediate
- decomposition of that and Ser side chain is LG but first His acts as acid to protonate Ser to make it better LG
- have enzyme and carboxylic acid
stabilising transition states
- once tetrahedral intermediate forms, O in C=O forms an oxyanion species to enter oxyanion hole and makes favourable H-bonds to the backbone NH groups of Gly193 and Ser195
- these favourable binding interactions reduce the activation energy
reaction and substrate specificity
-specificity of serine protease is determined by nature of S1 pocket and P1 residue of the substrate
Chymotrypsin specificity
S1 pocket favour big snug fit
P1 residue favour bulky side chain (F,Y,W)
-good VAW between lining of pocket and side chains
Trypsin specificity
S1 pocket favour -ve residue
P1 residue favours +ve side chain (K, R) so position -ve inside pocket (Asp189): electrostatic interactions
Elastase specificity
S1 pocket favours Val side chains that petrude into pocket, narrow it
P1 residue favours small, neutral side chains (G,A,V,S)
-snug fit, depth of pocket reduced
-blocked by Val190 and Val216 to allow ES complementarity
evolution of catalytic triad
- chymotrypsin, subtilisin, and serine carboxypeptidase II all have catalytic triad but are descended from DIFFERENT ancestral proteases: CONVERGENT evolution
- elastase, chymotrypsin, and trypsin= DIVERGENT evolution
Chymotrypsin ‘initial burst’ of product formation
- revealed by pre-steady state kinetics: refers to an experiment where you take an enzyme and you mix it in w/S and look at the first catalytic turnover
- burst phase: P is formed in equal amounts to E (mole basis)
- artificial substrate is ester: has Phe group to ensure recognition by chymotrypsin in S1 pocket
- hydrolysis to form c.acid + deprotonated alcohol in alkali pH
- yellow product can be measured using absorbent spectroscopy
- first initial burst of product to generate covalently attached acyl-enzyme intermediate; slower regeneration of enzyme us RDS catalytic cyle and limits catalysis in subsequent turnovers
similarity of catalytic mechanism to other proteases
- presence of Nu to attack carbonyl group
- presence of charges to polarise carbonyl group and stabilise tetrahedral intermediate
- presence of proton donor to make NH group better leaving group
