SERINEPROTEINASES

Gal·la AguinaliuJúlia Alonso

Berta Martínez

INDEX

• Introduction• General mechanism of action• Different topologies and foldings• Results• Conclusions• References

INTRODUCTION

Proteinases

• Proteinases catalyse the hydrolysis of covalent peptide bonds

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

• Groups:

SerineCysteineThreonine AsparticMetallo

• 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 identified1/3

Abundance measure of succes in evolutionary terms

These enzymes deserve attention

MEROPS classification

MEROPS database

• Clans: based on catalytic mechanism• Families: based on common ancestry

DegradomeDegradome: 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

SCOP classification

SCOP

Trypsin like

Prokaryotic proteinasesEukaryotic proteinases

Viral proteinases

Viral cysteine proteinase of trypsin fold

Trypsin

Elastase

Chymotrypsin

Subtisilin like

Subtilases

Serine-carboxyl proteinase

Trypsin-like: Zimogen activation

Enteropeptidase

Trypsinogen Trypsin

Proelastase Elastase Chymotrypsinogen Chymotrypsin

Zimogen: inactive enzyme precursor

Trypsin-like: Zimogen activation

Chymotrypsinogen activation

ChymotrypsinChymotrypsinogen

Ile 16

Asp 194

Chymotrypsinogen activation

ChymotrypsinChymotrypsinogen

Binding site

MECHANISM OF

ACTION

Four important structural features required for the catalitic action of SP:

1. Catalytic triad2. The oxanyon hole3. Polypeptide binding site4. Specificity pockets

Chemical mechanism of serine proteinases

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

Trypsin-like SP – Catalytic triad

Asp2

Ser2His2

The oxanyon hole (Gly193 and Ser195)

Cathalytic triad

Trypsin-like SP – The oxanyon hole

Trypsin-like SP – Substrate recognition site

Substrate recognition site

Trypsin-like SP – Specificity pocket

Trypsin Chymotrypsin

Elastase

Gly 226

Gly 216

Asp 189

Ser 189

Gly 216Gly 226

Thr 226

Val 216

Covalent bond formation

Tetrahedral transition state

Acyl-enzyme intermediate

Water activation

Tetrahedral transition state

Enzyme regeneration and product formation

Summary of the catalytic mechanism

Superimposition B-trypsin + Inhibitor 2AH4

InhibitorAsp102

His57

Ser195

• Inhibitor: 4-guanidinobenzoic acid

• Cyan: Beta-trypsin• Magenta: Beta-trypsin + Inhibitor• 2.233 A: distance betwen Ser195 and Inhibitor

Superimposition B-trypsin + Inhibitor 2AH4

Leupeptin inhibitor 2AGI

Leupeptin inhibitor 2AGI

His 57

Ser 195

Asp 102

Asp 189

TOPOLOGY and FOLDINGS

Trypsin-like 1FMG

• Evolution by gene duplication from a single ancestor proteinase domain

Chymotrypsinogen evolution, gene duplication

Trypsin-like 1FMG

• Trypsin– Fold: Trypsin-like serine proteinases

– Barrel– Greek-key – Duplication: consists of two domains of the same fold

Greek key Beta hairpin

123 4 5 6

• Subtilisin– 3 layers: a/b/a– Parallel beta-sheet of 7 strands – Left-handed crossover connection

between strands 2 & 3

Subtilisin-like 1ST3

• Subtilisin– 3 layers: a/b/a– Parallel beta-sheet of 7 strands – Left-handed crossover connection

between strands 2 & 3

Subtilisin-like 1ST3

• Subtilisin– 3 layers: a/b/a– Parallel beta-sheet of 7 strands – Left-handed crossover

connection between strands 2 & 3

Subtilisin-like 1ST3

C-terminal N-terminal

Prolyl oligopeptidase 1QFS

• N-terminal domain– Fold: 7-bladed beta-propeller

• Seven 4-stranded beta-sheet motifs

• Meander

Prolyl oligopeptidase 1QFS

• C-terminal domain– Fold: Alpha/beta-Hydrolases

• Core: 3 layers, a/b/a• Mixed beta-sheet of 8 strands• Strand 2 is antiparallel to the

rest

Prolyl oligopeptidase 1QFS

Clp peptidase 1YTF

• Clp peptidase– Fold: Clp/crotonase

– Core: 4 turns of beta (beta-beta-alpha)n superhelix

RESULTS

Chymotrypsins’ sequence alignment, CLUSTALW

Ser 195

Asp 102

His 57

Oxyanion hole

Main chain substrate binding

Trypsin-like enzymes’ sequence alignment, HMM

Ser 195

Asp 102

His 57

Oxyanion hole

Main chain substrate binding

Trypsin-like enzymes’ sequence alignment based on structure, STAMP

Ser 195

Asp 102

His 57

Oxyanion hole

Main chain substrate binding

Trypsins from different species’ superimposition

Sc 8.71RMS 1.27

• Streptomyces griseus, Trypsin• Sus scrofa, Beta Trypsin• Bos taurus, Trypsinogen

Trypsin-like enzymes’ superimposition

Sc 8.94RMS 1.02

• H. sapiens, plasma kallikrein• Sus scrofa, Beta Trypsin• H. sapiens, blood coagulation

factor XA

• Divergent evolution

Trypsin-like enzymes’ superimposition

Subtilisin-like enzymes’ sequence alignment, CLUSTAL

Ser 221

Asp 32

His 64

Oxyanion hole

Main chain substrate binding

Subtilisin-like enzymes’ sequence alignment, HMM

Ser 221

Asp 32

His 64

Oxyanion hole

Main chain substrate binding

Main chain substrate binding

Subtilisin-like enzymes’ alignment based on structure, STAMP

Ser 221

Asp 32

His 64

Oxyanion hole

Main chain substrate binding

Main chain substrate binding

Subtilisin-like enzymes’ superimposition

• D. nodosus, acidic extracel. subtilisin-like proteinase

• Vibrio sp., cold adapted subtilisin

• B. licheniformis, subtilisin carlsberg

Sc 7.82RMS 1.29

Serine proteinases’ superimposition

• H. sapiens, neutrophil elastase (trypsin-like)

• B. licheniformis, subtilisin carlsberg• A. sendaiensis, kumamolisin

apoenzyme (serine-carboxi peptidase)

Sc 1.48RMS 4.14

Trypsin-subtilisin superimposition

• Sus scrofa, Beta trypsin• B. licheniformis, subtilisin

carlsberg

Sc 0.54RMS 2.47

Similar catalytic triad, convergent evolution

SubtilisinTrypsin

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 His57 3.237 3.027

O2 of Asp102 and NHs His57 2.966 4.511

Asp2

Ser2

Asp2

Ser2

His2 His2

CONCLUSIONS

Conclusions

• Divergent evolution and gene duplication in trypsin-like enzymes• Convergent evolution between trypsin-like enzymes and subtilisin-like

enzymes • Different structure• Different sequence• Same mechanism of action

PROGRAMMES USED

• ClustalW• HMM• STAMP• XAM• Chimera• Rasmol

Programmes used

PDBProtein Species PDB

Subtilisin Bacillus lentus 3BX1, 1ST3Subtilisin Bacillus amyloliquefaciens 1SBTProlyl oligopeptidase Sus scrofa 1QFSClp peptidase Escherichia coli 1TYFPlasma kallikrein Homo sapiens 2ANWFactor XA Homo sapiens 1HCGBeta-trypsinogen Bos taurus 1TGNTrypsin Streptomyces griseus 1SGTSubtilisin Bacillus clausii 1MPTSubtilisin Savinase Bacillus lentus 1NDQSelenosubtilisin Bacillus subtilis 1SELThermitase (subtilisin-like serineproteinase)

Thermoactinomyces vulgaris 1THM

Mesentericopeptidase (subtilisin-like serine proteinase)

Bacillus pumilus 1MEE

Chymotrypsin inhibitor CI-2 Hordeum vulgare 2SNI

Protein Species PDB

Subtilisin-like proteinase APRV2

Dichelobacter nodosus 3LPC

Cold adapted subtilisin-like serine proteinase

Vibrio sp 1S2N

Subtilisin Carlsberg Bacillus licheniformis 1YU6

Extracellular subtilisin-like proteinase

Vibrio 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

REFERENCES

• 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/cr000033x

• Page, M. J., & Di Cera, E. (2008). Serine peptidases: Classification, structure and function. Cellular and Molecular Life Sciences, 65, 1220–1236. doi:10.1007/s00018-008-7565-9

• Polgár, 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

1. Which of these residues are part of the catalytic thriad in serine proteinases?a) Asp-Ser-Hisb) Asp-Thr-Hisc) Ser-Asp-Thrd) Ser-Gly-Hise) Asp-His-Thr

2. Proteinases are found in:a) Animalsb) Bacteria and plantsc) Archaea and virusesd) All of the answers above are incorrecte) a, b and c are correct

3. Regarding trypsin-like and subtilisin-like enzymes, they both have similar:f) Structureg) Sequenceh) Catalytic thriadi) Functionj) A, 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 5.5b) could probably have a SC value lower than 2c) could probably have a SC value between 5.5-9.8d) will probably have a RMSD value higher than 2e) will probably have a RMSD value higher than 5.5

5. Which different proteinases groups do exist?a) Serine and cysteinb) Serine, cystein, threonin and glycinec) Cystein, serine, threonin, aspartic and metallod) Cystein, metallo, serine, glycine and histidinee) All of the answers are incorrect

PEM

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 cleft

b) Catalytic triad, the oxyanion hole, the non-specificity pocket and the substrate binding sitec) Catalytic triad, the oxyanion hole, the specificity pocket and the substrate binding sited) Ser-Gly-His-Aspe) Asp-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 correct

8. Regarding serine proteinases tridimensional structure and folding:f) Trypsin-like enzymes have a left-handed crossover connectiong) Trypsin-like enzymes contain an anti-parallel betta sheet composed of 10 strandsh) Subtilisin-like enzymes contain a parallel betta sheet composed of 10 strandsi) Trypsin-like enzymes and subtilisin-like enzymes have similar structurej) Every answer above is incorrect

9. Which serine proteinase clan is most representative of the eukariotic proteome?a) PAb) SKc) SBd) SHe) 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 residuesb) Activation of trypsin to trypsinogen recquires a cleavage of 15-16 residuesc) Endopeptidases activate chymotrypsunogen into chymotrypsind) Elastase is synthetised as an already active enzyme in the duodenume) All answers are incorrect

PEM