SERINE PROTEINASES Gal·la - PowerPoint Presentation

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SERINEPROTEINASES

Gal·la AguinaliuJúlia AlonsoBerta Martínez

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INDEX

IntroductionGeneral mechanism of action

Different

topologies

and

foldings

Results

Conclusions

References

Slide3

INTRODUCTION

Slide4

P

roteinases Proteinases catalyse

the hydrolysis of covalent peptide bonds

Found in: Animals, plants, bacteria, archea and viruses

Groups:SerineCysteineThreonine AsparticMetallo

Slide5

Presence of a nucleophilic

serine residue at the active site of the

enzyme

Crucial roles in a

wide

variety of cellular and extracellular functions: blood clotting,

protein

digestion, cell signaling, inflammation and protein processing.

Introduction to serine proteinases

Of all

known

proteinases identified

1/3

Abundance

 measure of succes in evolutionary terms

T

hese enzymes deserve attention

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MEROPS classification

MEROPS database Clans: based on

catalytic

mechanism

Families

: based on common ancestry

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Degradome

Degradome: peptidases present within a genome

4

families

account

for over 40% of the human degradoma Ubiquitin- specific peptidases (CA, C19)

Zn-

dependent adamalysins (MA, M12)Prolyl oligopeptidases (SC, S9)

Trypsin-like serine peptidases (PA, S1, A)

PA



Eukariotic

SB, SC  Archea, prokaryotes,

plants

and fungi

Slide8

SCOP classification

SCOP

Trypsin

like

Prokaryotic

proteinases

Eukaryotic

proteinasesViral

proteinases

Viral

cysteine

proteinase of trypsin foldTrypsin

Elastase

Chymotrypsin

Subtisilin

like

Subtilases

Serine-carboxyl proteinase

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Trypsin-like

: Zimogen activation

Enteropeptidase

Trypsinogen

Trypsin

Proelastase

Elastase

Chymotrypsinogen

Chymotrypsin

Zimogen:

inactive

enzyme

precursor

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Trypsin-like: Zimogen activation

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Chymotrypsinogen

activation

Chymotrypsin

Chymotrypsinogen

Ile

16

Asp 194

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Chymotrypsinogen

activation

Chymotrypsin

Chymotrypsinogen

B

inding

site

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MECHANISM OF

ACTION

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Four important structural

features required for the catalitic action of SP:

Catalytic

triad

The

oxanyon

holePolypeptide binding siteSpecificity pockets

Chemical

mechanism

of serine proteinases

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The catalytic triad

spans the active site cleft, with Ser195

on

one

side

and

Asp102 and His57 on the other.

Trypsin-like

SP – Catalytic

triad

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Trypsin-like

SP – Catalytic triad

Asp

2

Ser

2

His

2

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The

oxanyon hole (Gly193 and Ser195)

Cathalytic

triad

Trypsin-like

SP – The

oxanyon

hole

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Trypsin-like

SP – Substrate recognition site

Substrate

recognition

site

Slide19

Trypsin-like

SP – Specificity pocket

Trypsin

Chymotrypsin

Elastase

Gly

226

Gly

216

Asp 189

Ser 189

Gly

216

Gly

226

Thr

226

Val 216

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Covalent

bond formation

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Tetrahedral

transition state

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Acyl-enzyme

intermediate

Slide23

Water

activation

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Tetrahedral

transition state

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Enzyme

regeneration and product

formation

Slide26

Summary

of the

catalytic

mechanism

Slide27

Superimposition

B-trypsin + Inhibitor 2AH4

Inhibitor

Asp102

His57

Ser195

Inhibitor

:

4-guanidinobenzoic

acid

Cyan

: Beta-

trypsin

Magenta: Beta-trypsin + Inhibitor 2.233 A: d

istance

betwen Ser195 and

Inhibitor

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Superimposition

B-trypsin + Inhibitor 2AH4

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Leupeptin

inhibitor 2AGI

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Leupeptin

inhibitor 2AGI

His

57

Ser 195

Asp 102

Asp 189

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TOPOLOGY and FOLDINGS

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Trypsin-like

1FMG

Slide33

Evolution by

gene duplication from a single ancestor proteinase

domain

Chymotrypsinogen

evolution,

gene

duplication

Slide34

Trypsin-like 1FMG

Trypsin

Fold

:

Tryp

sin-like

serine

proteinases

BarrelGreek-key

Duplication

:

consists of two domains of

the

same

fold

Greek

key

Beta

hairpin

1

2

3

4

5

6

Slide35

 Subtilisin3 layers:

a/b/aParallel beta-sheet of 7 strands Left-handed crossover connection between strands 2 & 3

Subtilisin-like

1ST3

Slide36

 

Subtilisin

3 layers: a/b/a

Parallel beta-sheet of 7 strands

Left-handed crossover connection between strands 2 & 3

Subtilisin-like

1ST3

Slide37

 

Subtilisin

3 layers: a/b/a

Parallel beta-sheet of 7 strands

Left-handed crossover connection between strands 2 & 3

Subtilisin-like

1ST3

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C-terminal

N-terminal

Prolyl

oligopeptidase

1QFS

Slide39

N-terminal

domain

Fold

: 7-bladed

beta-propeller

Seven 4-stranded

beta-sheet

motifs

Meander

Prolyl

oligopeptidase

1QFS

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C-terminal

domain

Fold

:

Alpha

/

beta-HydrolasesCore: 3 layers, a/b/aMixed

beta-sheet of 8 strandsStrand 2 is antiparallel

to the rest

Prolyl

oligopeptidase

1QFS

Slide41

Clp peptidase

1YTF

 

Clp

peptidase

Fold:

Clp

/

crotonase

Core: 4 turns

of beta (beta-beta-alpha)n

superhelix

Slide42

RESULTS

Slide43

Chymotrypsins

’ sequence alignment, CLUSTALW

Ser 195

Asp 102

His

57

Oxyanion hole

Main chain

substrate binding

Slide44

Trypsin-like

enzymes’ sequence alignment, HMM

Ser 195

Asp 102

His

57

Oxyanion hole

Main chain

substrate binding

Slide45

Trypsin-like

enzymes’ sequence alignment

based

on

structure

, STAMP

Ser 195

Asp 102

His

57

Oxyanion

hole

Main chain substrate binding

Slide46

Trypsins from

different species’ superimposition

Sc

8.71

RMS

1.27

Streptomyces

griseus

,

Trypsin

Sus

scrofa

, Beta

Trypsin

Bos

taurus

,

Trypsinogen

Slide47

Trypsin-like enzymes

’ superimposition

Sc

8.94

RMS

1.02

H. sapiens

, plasma

kallikrein

Sus

scrofa

, Beta

Trypsin

H. sapiens

,

blood

coagulation

factor XA

Slide48

Divergent evolution

Trypsin-like enzymes’ superimposition

Slide49

Subtilisin-like

enzymes’ sequence alignment, CLUSTAL

Ser 221

Asp 32

His

64

Oxyanion hole

Main

chain substrate binding

Slide50

Subtilisin-like

enzymes’ sequence alignment, HMM

Ser 221

Asp 32

His

64

Oxyanion hole

Main chain

substrate binding Main chain

substrate binding

Slide51

Subtilisin-like

enzymes’ alignment based on

structure

, STAMP

Ser 221

Asp 32

His

64

Oxyanion

hole

Main chain substrate binding Main

chain substrate

binding

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Subtilisin-like enzymes

’ superimposition

D.

nodosus

,

acidic

extracel

.

subtilisin

-

like

proteinase

Vibrio

sp.

,

cold adapted subtilisin

B.

licheniformis, subtilisin carlsberg

Sc

7.82

RMS

1.29

Slide53

Serine

proteinases’ superimposition

H. sapiens,

neutrophil

elastase

(

trypsin-like

)

B.

licheniformis

,

subtilisin

carlsberg

A.

sendaiensis

,

kumamolisin apoenzyme (

serine-carboxi

peptidase)Sc

1.48

RMS

4.14

Slide54

Trypsin-subtilisin superimposition

Sus

scrofa

,

Beta

trypsin

B.

licheniformis

,

subtilisin

carlsberg

Sc

0.54

RMS

2.47

Slide55

Similar

catalytic triad, convergent evolution

Subtilisin

Trypsin

Hydrogen

bonds

Tryspin

(

Distances A)

Subtilisin (Distances (A)N1-H of His57 and O1 of Asp102

2.739

2.839

OH of Ser195

and N2-H of His573.2373.027O2 of Asp102 and NHs His57

2.966

4.511

Asp2

Ser2Asp2Ser2

His2

His2

Slide56

CONCLUSIONS

Slide57

Conclusions

Divergent

evolution

and gene

duplication

in

trypsin-like enzymesConvergent

evolution between trypsin-like enzymes

and subtilisin-like enzymes Different

structure

Different

sequenceSame mechanism of action

Slide58

PROGRAMMES USED

Slide59

ClustalWHMMSTAMPXAMChimera

Rasmol

Programmes

used

Slide60

PDB

ProteinSpecies

PDB

Subtilisin

Bacillus

lentus

3BX1, 1ST3

Subtilisin

Bacillus

amyloliquefaciens

1SBT

Prolyl

oligopeptidaseSus scrofa

1QFS

Clp

peptidase

Escherichia coli1TYFPlasma kallikreinHomo sapiens2ANW

Factor XA

Homo sapiens1HCGBeta-trypsinogenBos taurus

1TGN

Trypsin

Streptomyces

griseus

1SGT

Subtilisin

Bacillus

clausii

1MPT

Subtilisin

Savinase

Bacillus

lentus

1NDQ

Selenosubtilisin

Bacillus

subtilis

1SEL

Thermitase

(

subtilisin-like

serineproteinase

)

Thermoactinomyces

vulgaris

1THM

Mesentericopeptidase

(

subtilisin-like

serine

proteinase

)

Bacillus

pumilus

1MEE

Chymotrypsin

inhibitor

CI-2

Hordeum

vulgare

2SNI

Slide61

Protein

SpeciesPDB

Subtilisin-like

proteinase

APRV2

Dichelobacter

nodosus

3LPC

Cold adapted subtilisin-like

serine

proteinase

Vibrio

sp1S2NSubtilisin

Carlsberg

Bacillus

licheniformis1YU6

Extracellular subtilisin-like proteinaseVibrio sp.1SH7

serine-carboxyl proteinase

Pseudomonas sp.1GA6

Kumamolisin

-As (

serine

-carboxil

proteinase

)

Alicyclobacillus

sendaiensis

1SN7

Kumamolisin

Bacillus

sp

.

1T1G

Neutrophil

elastase

Homo sapiens

3Q76

Chymotrypsinogen

A

Bos

taurus

1EX3

Gamma-Chymotrypsin

A

Bos

taurus

1GMC

Cationic

trypsin

Bos

taurus

4I8G

Beta-

Trypsin

Sus

scrofa

1FMG

Elastase

Sus

scrofa

1C1M

Chymotrypsin

Bos

taurus

1GMC, 2CHA

PDB

Slide62

REFERENCES

Slide63

Di Cera, E. (2009). Serine proteases

. IUBMB Life, 61(May), 510–515. doi:10.1002/iub.186

Hedstrom

, L. (2002).

Serine

protease

mechanism and specificity. Chemical

Reviews

, 102, 4501–4523. doi:10.1021/cr000033xPage, M. J., & Di Cera, E. (2008). Serine peptidases

: Classification, structure and function. Cellular

and Molecular Life

Sciences

,

65, 1220–1236. doi:10.1007/s00018-008-7565-9Polgár, L. (2005). The catalytic triad of serine

peptidases

. Cellular and Molecular Life

Sciences, 62, 2161–2172. doi:10.1007/s00018-005-5160-x

Branden & Tooze (1998), Introduction to protein structure, 2nd ed.W. Pratt & Cornely

(2013), Essential Biochemistry

, 3rd ed.References

Slide64

1. Which of these

residues are part of the catalytic thriad in serine

proteinases

?

a)

Asp-Ser-His

b) Asp-Thr-Hisc) Ser-Asp-Thrd) Ser-Gly-HisAsp-His-Thr

2.

Proteinases are found in:a) Animalsb) Bacteria and plants

c) Archaea and virusesd) All of the answers above are incorrecte) a, b and c are correct3. Regarding trypsin-like and subtilisin

-like enzymes, they both have similar:

StructureSequence

Catalytic thriad

FunctionA, b, c and d are true 4. A superimposition with STAMP of different chymotrypsins from different species… a) could probably have a SC value lower than 5.5 b) could probably have a SC value lower than 2 c) could probably have a SC value between 5.5-9.8

d) will probably have a RMSD value higher than 2

e) will probably have a RMSD value higher than 5.5 5. Which different proteinases groups do exist?

a) Serine and cystein

b) Serine, cystein, threonin and glycine c) Cystein, serine, threonin, aspartic and metallo d) Cystein, metallo, serine, glycine and histidine e) All of the answers are incorrect

PEM

Slide65

6. Which

are the four important features in serine proteinases?

a)

the

oxyanion hole, the non-specificity pocket, the catalytic triad,

and

the substrate binding cleftb) Catalytic triad

, the oxyanion hole, the non-specificity pocket and

the substrate

binding

site

c) Catalytic triad, the oxyanion hole, the specificity pocket

and the

substrate binding sited)

Ser-Gly-His-AspAsp-His-Thr-Ser

7. Evolution processes in trypsin like enzymes and in subtilisin-like enzymes:a) Divergent evolution and gene duplication in substilisin-like enzymesb) Convergent evolution in different

trypsin-like enzymesc) Gene duplication in subtilisin-like enzymesd) Divergent evolution in

trypsin-like enzymes and convergent evolution between trypsin-like enzymes and subtilisin-like enzymes e) a, b and c are correct8. Regarding serine proteinases

tridimensional structure and folding:

Trypsin

-like enzymes have a left-handed crossover connection

Trypsin-like enzymes contain an anti-parallel

betta

sheet composed of 10 strands

Subtilisin

-like enzymes contain a parallel

betta

sheet composed of 10 strands

Trypsin

-like enzymes and

subtilisin

-like enzymes have similar structure

Every answer above is incorrect

9. Which serine

proteinase

clan is most representative of the

eukariotic

proteome?

a) PA

b) SK

c) SB

d) SH

e) SJ

10. About

zimogen

activation in

trypsin

-like serine

proteinases

, which answer is correct:

a)

Activation of

trypsinogen

to

trypsin

recquires

a cleavage of 15-16 residues

b) Activation of

trypsin

to

trypsinogen

recquires

a cleavage of 15-16 residues

c)

Endopeptidases

activate

chymotrypsunogen

into

chymotrypsin

d)

Elastase

is

synthetised

as an already active enzyme in the duodenum

e) All answers are incorrect

PEM