SERINE
PROTEINASES
Gal·la Aguinaliu
Júlia 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:
Serine
Cysteine
Threonine
Aspartic
Metallo
Introduction to serine proteinases
• 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.
1/3
Of all known proteinases identified
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
Degradome
Degradome: peptidases present within a genome
PA  Eukariotic
4 families account for over 40% of the human degradoma
SB, SC  Archea,
prokaryotes, plants and
fungi
•
•
•
•
Ubiquitin- specific peptidases (CA, C19)
Zn- dependent adamalysins (MA, M12)
Prolyl oligopeptidases (SC, S9)
Trypsin-like serine peptidases (PA, S1, A)
SCOP classification
SCOP
Prokaryotic
proteinases
Eukaryotic proteinases
Trypsin like
Viral proteinases
Viral cysteine
proteinase of trypsin
fold
Subtilases
Subtisilin like
Serine-carboxyl
proteinase
Trypsin
Chymotrypsin
Elastase
Trypsin-like: Zimogen activation
Zimogen: inactive enzyme precursor
Enteropeptidase
Trypsinogen
Proelastase
Elastase
Trypsin
Chymotrypsinogen
Chymotrypsin
Trypsin-like: Zimogen activation
Chymotrypsinogen activation
Chymotrypsin
Chymotrypsinogen
Asp 194
Ile 16
Chymotrypsinogen activation
Chymotrypsin
Chymotrypsinogen
Binding site
MECHANISM
OF
ACTION
Chemical mechanism of serine proteinases
Four important structural features required
for the catalitic action of SP:
1.
2.
3.
4.
Catalytic triad
The oxanyon hole
Polypeptide binding site
Specificity pockets
Trypsin-like SP – Catalytic triad
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
His2
Asp2
Ser2
Trypsin-like SP – The oxanyon hole
The oxanyon hole
(Gly193 and Ser195)
Cathalytic triad
Trypsin-like SP – Substrate recognition site
Substrate recognition site
Trypsin-like SP – Specificity pocket
Gly 216
Gly 226
Gly 226
Gly 216
Asp 189
Ser 189
Chymotrypsin
Trypsin
Thr 226
Val 216
Elastase
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
Ser195
His57
Asp102
Inhibitor
• Cyan: Beta-trypsin
• Inhibitor: 4-guanidinobenzoic
• Magenta: Beta-trypsin + Inhibitor
acid
• 2.233 A: distance betwen Ser195 and Inhibitor
Superimposition B-trypsin + Inhibitor
2AH4
Leupeptin inhibitor
2AGI
Leupeptin inhibitor
His
57
2AGI
Asp
102
Ser
195
Asp
189
TOPOLOGY and
FOLDINGS
Trypsin-like
1FMG
Chymotrypsinogen evolution, gene duplication
• Evolution by gene duplication from a single ancestor proteinase domain
Trypsin-like
1FMG
Greek key
3
2
1
Beta hairpin
4
5
6
• Trypsin
– Fold: Trypsin-like serine proteinases
– Barrel
– Greek-key
– Duplication: consists of two domains of the
same fold
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
•
Subtilisin
– 3 layers: a/b/a
– Parallel beta-sheet of 7 strands
– Left-handed crossover
connection between strands 2
&3
Prolyl oligopeptidase
C-terminal
1QFS
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
Clp peptidase
•
Clp peptidase
– Fold: Clp/crotonase
– Core: 4 turns of beta (beta-beta-alpha)n
superhelix
1YTF
RESULTS
Chymotrypsins’ sequence alignment, CLUSTALW
His 57
Asp 102
Oxyanion hole
Main chain substrate binding
Ser 195
Trypsin-like enzymes’ sequence alignment, HMM
His 57
Asp 102
Ser 195
Oxyanion hole
Main chain substrate binding
Trypsin-like enzymes’ sequence alignment based on structure, STAMP
His 57
Asp 102
Ser 195
Oxyanion hole
Main chain substrate binding
Trypsins from different species’ superimposition
• Streptomyces griseus, Trypsin
• Sus scrofa, Beta Trypsin
• Bos taurus, Trypsinogen
Sc 8.71
RMS 1.27
Trypsin-like enzymes’ superimposition
• H. sapiens, plasma kallikrein
• Sus scrofa, Beta Trypsin
• H. sapiens, blood coagulation
factor XA
Sc 8.94
RMS 1.02
Trypsin-like enzymes’ superimposition
• Divergent evolution
Subtilisin-like enzymes’ sequence alignment, CLUSTAL
Asp 32
His 64
Oxyanion hole
Main chain substrate binding
Ser 221
Subtilisin-like enzymes’ sequence alignment, HMM
Asp 32
His 64
Main chain substrate binding
Oxyanion hole
Main chain substrate binding
Ser 221
Subtilisin-like enzymes’ alignment based on structure, STAMP
Asp 32
His 64
Main chain substrate binding
Oxyanion hole
Main chain substrate binding
Ser 221
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
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
Trypsin-subtilisin superimposition
• Sus scrofa, Beta trypsin
• B. licheniformis, subtilisin
carlsberg
Sc 0.54
RMS 2.47
Similar catalytic triad, convergent evolution
Trypsin
Subtilisin
His2
His2
Ser2
Ser2
Asp2
Asp2
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
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
Programmes used
•
•
•
•
•
•
ClustalW
HMM
STAMP
XAM
Chimera
Rasmol
PDB
Protein
Species
PDB
Subtilisin
Bacillus lentus
3BX1, 1ST3
Subtilisin
Bacillus amyloliquefaciens
1SBT
Sus scrofa
1QFS
Clp peptidase
Escherichia coli
1TYF
Plasma kallikrein
Homo sapiens
2ANW
Factor XA
Homo sapiens
1HCG
Bos taurus
1TGN
Streptomyces griseus
1SGT
Subtilisin
Bacillus clausii
1MPT
Subtilisin Savinase
Bacillus lentus
1NDQ
Selenosubtilisin
Bacillus subtilis
1SEL
Thermoactinomyces vulgaris
1THM
Mesentericopeptidase (subtilisinlike serine proteinase)
Bacillus pumilus
1MEE
Chymotrypsin inhibitor CI-2
Hordeum vulgare
2SNI
Prolyl oligopeptidase
Beta-trypsinogen
Trypsin
Thermitase (subtilisin-like
serineproteinase)
PDB
Protein
Species
PDB
Dichelobacter nodosus
3LPC
Vibrio sp
1S2N
Bacillus licheniformis
1YU6
Extracellular subtilisin-like
proteinase
Vibrio sp.
1SH7
serine-carboxyl proteinase
Pseudomonas sp.
1GA6
Alicyclobacillus sendaiensis
1SN7
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
Subtilisin-like proteinase
APRV2
Cold adapted subtilisin-like
serine proteinase
Subtilisin Carlsberg
Kumamolisin-As (serinecarboxil proteinase)
Kumamolisin
REFERENCES
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.
PEM
1. Which of these residues are part of the catalytic thriad in serine proteinases?
a)
Asp-Ser-His
b)
Asp-Thr-His
c)
Ser-Asp-Thr
d)
Ser-Gly-His
e)
Asp-His-Thr
2. Proteinases are found in:
a)
Animals
b)
Bacteria and plants
c)
Archaea and viruses
d)
All of the answers above are incorrect
e)
a, b and c are correct
3. Regarding trypsin-like and subtilisin-like enzymes, they both have similar:
a)
Structure
b)
Sequence
c)
Catalytic thriad
d)
Function
e)
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 5.5
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
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 site
c)
Catalytic triad, the oxyanion hole, the specificity pocket and the substrate binding site
d)
Ser-Gly-His-Asp
e)
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 enzymes
b)
Convergent evolution in different trypsin-like enzymes
c)
Gene duplication in subtilisin-like enzymes
d)
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:
a)
Trypsin-like enzymes have a left-handed crossover connection
b)
Trypsin-like enzymes contain an anti-parallel betta sheet composed of 10 strands
c)
Subtilisin-like enzymes contain a parallel betta sheet composed of 10 strands
d)
Trypsin-like enzymes and subtilisin-like enzymes have similar structure
e)
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