Chymotrypsin

What are the functions of chymotrypsins

digestion, immune response, blood coagulation, reproduction

what are the two families of serine proteases?

chymotrypsin-like and substilisin-like

what is a serine protease?

enzymes that cleave peptide bonds in proteins, in which serine serves as nucleophilic amino acid at enzyme active site

How have substilisin-like serine proteases evolved?

convergently. Since structurally and sequence very different even though same catalytic triad

what amino acids are the in catalytic triad?

glutamine, histidine and serine

where does chymotrypsin cleave peptides?

C-terminus of aromatic Amino acids

What can chymotrypsin cleave in the binding pocket of the enzyme?

bulky aromatics (W, Y, F, L, M)

What can trypsin cleave in binding pocket of the enzyme?

positively charged (Lys, and Arg)

What can elastase cleave in binding pocket of the enzyme?

small non-charged molecules (Ala, Val, Thr)

what can the R group on amino acid next to one cleaved not be?

proline

What is the first step of reaction mechanism of chymotrypsin?

substrate attaches to enzymes at main chain binding site and specificity pocket with scissile bond expose to tried (ES formation)

What is second step of reaction mechanism of chymotrypsin?

covalent bond formation between-C=O and S195 hydroxyl, tetrahedral intermediate as TS, oxyanion hole stabilisation, H57 binds the released H+ from S195 (ES*)

What is third step of reaction mechanism of chymotrypsin?

Acyl-enzyme intermediate and peptide bond hydrolysis peptide with new N-term released from enzyme taking -H from H57. (EP*)

What is fourth step of reaction mechanism of chymotrypsin?

water molecule placed next to H57 and acyl-enzyme intermediate at S195 (EP*)

What is fifth step of reaction mechanism of chymotrypsin?

water molecule initiates nu- attack and undergoes reaction with one -H attaching to H57 and -OH to acyl-enzyme intermediate forming again tetrahedral TS of new C-terminal peptide fragment (EP*)

What is sixth step of reaction mechanism of chymotrypsin?

Peptide released while H57 donates -H (from water molecule) to S195 (E+P)

What does n-actyl-l-phenylalanine p-nitrophenyl yield on cleavage and what colour is it?

p-nitrophenolate (yellow)

What are the uses of serine proteases now?

coagulation, immune system, complement system (inflammation, thrombosis)

How have serine proteases divergently evolved

used to just be digestive enzymes, now blood clotting ,inflammation, immune system

what other protease likely evolved from the same ancestral protease as serine?

cysteine protease

what is the structure of serine protease?

two beta barrels

Why could swapping for cysteine be a benefit for proteases?

don't need the aspartate in catalytic triad since cysteine is a much better acid

What does the evolution of serine proteases tell us?

catalytic triad of Ser-His-Asp very important

What are serpins?

serine protease inhibitors and other functions such as storage, hormone carriage proteins, tumour suppressor genes

What serpins are used for blood clotting?

anti-thrombin, heparin co-factor II

What serpins are used for inflammation?

anti-trypsin, anti-chymotrypsin, C1-inhibitor

What serpins are used for immune system?

C1-inhibitor

What serpins are used for tissue remodelling?

Plasminogen activator inhibitor-1

how does C1-inhibitor work?

inhibits HMW-kinongen to bradykinin and inhibits formation of C2C4 complex

How do serpins work?

Reactive centre loop is attacked by acyl-enzyme intermediate which causes conformation change with catalytic triad out of position so it cannot cleave

What happens when serpins go from stressed to relaxed state?

protease binds to the reactive centre loop and this is cleaved and inserts into the A-sheet which deforms and inhibits protease

With thrombin how can you get regulation?

anti-thrombin binds heparin to expose arginine better, thrombin binds and then is inactivated with heparin released

How does heparin change anti-thrombin?

extends helix D and forms P helix, in binding site, expelling P14 serine residue of serpin RCL initially buried

How can you control serpins that are latent (AKA don't spontaneously convert from R->S)

through cofactor binding that controls the C-sheet to peel and allow full RCL insertion

What are serpin diseases(serinopathies)?

mutations in serpins that result in protein misfielding and formation of inactive long chain polymers (dimer, trimers and more).

Name two serpins used for transport not for proteases

thyroxine binding globulin and cortisol binding protein

Name a serpin used purely as storage protein

ovalbumin

What serpin is a cofactor in blood clotting

Protein Z

What serpin is a regulator in Drosophila development

Scarface

Name organophosphorous serine proteases inhibitors

sarin, VX, novichok

How do organophosphorus inhibit serine proteases?

react with oxygen on serine, and then coordinate to form an oxyanion hole, so it cannot be cleaved by histidine.

What are the four classes of proteases?

serine, cysteine, aspartyl and metalloproteases

How do serine and cysteine protease active sites work

the characterising residue is usually paired with a proton-withdrawing group to promote nucleophilic attack on the peptide bond

How does the activation of serine and cysteine differ from aspartyl and metalloproteases?

aspartyl and metalloproteases activate a water molecule to serve as a nucleophile rather than using a functional group of the enzyme itself