Caracterizarea enzimei

Enzyme characterization

Molecule gigant Masă molec. 5-5000 KDa

(20-100 KDa)

Giant molecules Molecular weight 5-5000 KDa

(20-100 KDa)

Este structura extrem de complexă?

Is it an enzyme characterized by a

bewildering complex structure?

Enzimele Familia proteinelor

Enzymes Family of proteins

Structura Secvența liniară de AA Legaturi

amidice

Structure Linear sequence of AA Amide

linkage

Impachetare3D unica

“fold”a unique 3D

Caracterizarea enzimei

Enzyme characterization

Portiune din partea N-terminala a unei polipeptide

A portion of N-terminal end of a polypeptide chain

Structura

primara

3D

Activitatea

catalitica

The primary

structure

3D

Catalitic

activity

Zhang, W.-B. et al. (2017) Materials Horizons, 4(2), 117–132

Caracterizarea enzimei

Enzyme characterization

Determinarea structurii primare Determination of primary structure

Pehr Edman

www.qventas.com

Degradarea

EdmanEdman

degradation

Cuplarea

cu FITC

PITC

coupling

Clivarea

AA terminal

Incalzire/H+

PITC-AA1

is cleaved

heat/H+

Derivat FTH

cromatografie

PTH derivate

chromatography

Scindarea proteolitică Proteolytic cleavageProteaza Protease Natura legaturii peptidice clivate

Peptide bond selectivity

N-terminal -----XYZ----------C-terminal

X Y Z

Tripsina

Clostripaina

Chimotripsina

Elastaza

Papaina

Endoproteaza

Glu-C

Pepsina

Trypsin

Clostripain

Chymotrypsin

Elastase

Papain

Glu-C

Endoprotease

Pepsin

-

-

-

-

Phe

-

-

Lys, Arg

Arg

Trp, Tyr, Phe

Leu, Met, Ala

Lys

Glu, Asp

Phe

-

-

-

-

Leu si (and) Trp

-

Phe, Trp, Tyr

Caracterizarea enzimei

Enzyme characterization

Determinarea structurii primare Determination of primary structure

https://www.mpi-magdeburg.mpg.de/

CLASA Caracteristici inhibitor exemple functia

Serin proteaze situsul activ fluorofosfat tripsina digestia

H57, D102, S195 trombina coagulare sange

plasmina scindare cheaguri

subtilisina digestia

Cistein proteaze situsul activ iodoacetat papaina digestia

C25, H159, N175 strept. proteinaza digestia

catepsin B digestia

Acid proteaze pH optim acid diazocetone pepsina digestia

D32, D215 chimozina coagulare lapte

Metaloproteaze Zn2+, E270 o-phenanthroline carboxipeptidazele digestia

Zn2+, Ca2+ o-phenanthroline termolizina digestia

E143, H231

Scindarea proteolitică Proteolytic cleavage

Caracterizarea enzimei

Enzyme characterization

Determinarea structurii primare Determination of primary structure

CLASS Characteristics inhibitor Example Function

Serin protease activ site fluorophosphate trypsin digestion

H57, D102, S195 thrombin thrombosis

plasmin Converts fibrin

into soluble products

subtilisine digestion

Cystein protease activ site iodoacetate papain digestion

C25, H159, N175 strept. proteinase digestion

cathepsin B digestion

Acid proteases optimal acidic pH diazoketones pepsin digestion

D32, D215 chymosin

renin milk coagulation

Metaloproteases Zn2+, E270 o-phenanthroline carboxipeptidases digestion

Zn2+, Ca2+ o-phenanthroline thermolysin digestion

E143, H231

Scindarea proteolitică Proteolytic cleavage

Caracterizarea enzimei

Enzyme characterization

Determinarea structurii primare Determination of primary structure

Tripsina-scindeaza la legaturile peptidice Lys(Arg)-X

Scindarea proteolitică Proteolytic cleavage

Caracterizarea enzimei

Enzyme characterization

Determinarea structurii primare Determination of primary structure

Trypsin-cleaves peptide bonds Lys(Arg)-X

Schwartz. et al. (2014) J Anal Atom Spectrom

Scindarea metioninei Methionine cleavage

Caracterizarea enzimei

Enzyme characterization

Determinarea structurii primare Determination of primary structure

Scindare chimică Chemical cleavage

www.chemistry.iitkgp.ac.in

Enzima

puraReactii de

clivare

Etapa 1 Amestec de

fragmenteSepararea

fragmentelor

Etapa 2 Fragmente

purificate

Etapa 3

secventierea

Set de

secvente

individuale

Etapa 4

-clivarea

alternativa;

-separarea si

secventiereaSecventa

completa

Amestec

complexProteoliza

PeptideSepararea

fragmentelor

HPLC Fragmente

purificate

LC-MS/MS

Izolare

peptidaFragmentare

(Spectru de masa)

Tripsina

Baza de date

Secvente

Complete

Caracterizarea enzimei

Enzyme characterization

Determinarea structurii primare Determination of primary structure

Pure

EnzymeCleavage

reaction

Step 1 Mixture of

fragmentsFragments

separation

Step 2 Purified

fragments

Step 3

sequencing

Set of

individual

sequences

Step 4

-alternative

cleavage;

-separation and

sequencingFull

sequence

Complex

mixtureProteolysis

PeptidesSeparation

HPLC Purified

fragments

LC-MS/MS

Peptide

IsolationFragmentation

(mass spectrum)

Trypsin

Database

Full

sequence

Caracterizarea enzimei

Enzyme characterization

Determinarea structurii primare Determination of primary structure

Structura

primara

Masa moleculara

Localizare AA

Identificarea de parti

functionale

Fosfolipaza A2ALWQF

interacțiuni cu

substratul

nepolarStabilirea

relatii de evolutie

pI

Caracterizarea enzimei

Enzyme characterization

Determinarea structurii primare Determination of primary structure

Primary

structure

Mw

Locate a particulate AA

Identifying of functional

parts of

Phospholipase A2ALWQF

interactions with

nonpolar

substrateTo explore evolutionary

relationships

between enzymes

pI

β-D-galactozidaza (E Coli)

1021 AA

fosforilaza (muschi iepure)

841 AA

β-D-galactosidase (E Coli)

1021 AA

phosphorilase (rabbit muscle)

841 AA

Structura primară Estimarea Masa moleculara

NCBI http://www.ncbi.nlm.nih.gov/pubmed

Cea mai mare baza de date

UniProt http://www.uniprot.org/

P0DJD7.1 Pepsina umana 1-388

MKWLLLLGLVALSECIMYKVPLIRKKSLRRTLSERGLLKDFLKKHNLNP

ARKYFPQWEAPTLVDEQPLENYLDMEYFGTIGIGTPAQDFTVVFDTGS

SNLWVPSVYCSSLACTNHNRFNPEDSSTYQSTSETVSITYGTGSMTG

ILGYDTVQVGGISDTNQIFGLSETEPGSFLYYAPFDGILGLAYPSISSSGA

TPVFDNIWNQGLVSQDLFSVYLSADDQSGSVVIFGGIDSSYYTGSLNW

VPVTVEGYWQITVDSITMNGEAIACAEGCQAIVDTGTSLLTGPTSPIANI

QSDIGAS ENSDGDMVVSCSAISSLPDIVFTINGVQYPVPPSAYILQSEG

SCISGFQGMNLPTESGELWILGDVFIRQYFTVFDRANNQVGLAPVA

Caracterizarea enzimei

Enzyme characterization

Determinarea structurii primare Determination of primary structure

Primary structure Estimation of Mw

http://web.expasy.org/peptide_mass/

Mw (masa monoizotopica): 41950.55

Structura primară Estimarea Masa moleculara

Caracterizarea enzimei

Enzyme characterization

Determinarea structurii primare Determination of primary structure

Primary structure Estimation of Mw

https://web.expasy.org/compute_pi/

Structura primară Estimarea punctului izoelectric

Caracterizarea enzimei

Enzyme characterization

Determinarea structurii primare Determination of primary structure

Primary structure Estimation of isoelectric point

http://isoelectric.org/

Mw: 57304.81pI: 5.89

Fosfatază alcalină umană

human alkaline phosphatase

Structura primară Estimarea punctului izoelectric

Caracterizarea enzimei

Enzyme characterization

Determinarea structurii primare Determination of primary structure

Primary structure Estimation of isoelectric point

http://protcalc.sourceforge.net/cgi-bin/protcalc

http://www.biosyn.com/PeptidePropertyCalculator/

Mw: 57307.72pI: 6.35

Fosfatază alcalină umană

pI: 6.67

www.bioinformatics.org/sms2/protein_iep.html

pI: 6.66

Fosfatază alcalină umană

human alkaline phosphatase

Structura primară Estimarea punctului izoelectric

Caracterizarea enzimei

Enzyme characterization

Determinarea structurii primare Determination of primary structure

Primary structure Estimation of isoelectric point

MKWLLLLGLVALSECIMYKVPLIRKKSLRRTLSERGLLKDFLKKHNLNP

ARKYFPQWEAPTLVDEQPLENYLDMEYFGTIGIGTPAQDFTVVFDTGS

SNLWVPSVYCSSLACTNHNRFNPEDSSTYQSTSETVSITYGTGSMTG

ILGYDTVQVGGISDTNQIFGLSETEPGSFLYYAPFDGILGLAYPSISSSGA

TPVFDNIWNQGLVSQDLFSVYLSADDQSGSVVIFGGIDSSYYTGSLNW

VPVTVEGYWQITVDSITMNGEAIACAEGCQAIVDTGTSLLTGPTSPIANI

QSDIGAS ENSDGDMVVSCSAISSLPDIVFTINGVQYPVPPSAYILQSEG

SCISGFQGMNLPTESGELWILGDVFIRQYFTVFDRANNQVGLAPVA

Secventa semnal / Signal sequence

http://www.signalpeptide.de/index.php

Peptida activatoare / Activation peptide

Structura primară Secvența semnal a enzimei

Caracterizarea enzimei

Enzyme characterization

Determinarea structurii primare Determination of primary structure

Primary structure Enzyme signal sequence

Structura primara Locatie enzima Secventa semnal

Kalderon, B. & Pines, O. (2014),

Protein folding as a driving force for dual protein

targeting in eukaryotes,

Front. Mol. Biosci.,1, 1-9

Caracterizarea enzimei

Enzyme characterization

Determinarea structurii primare Determination of primary structure

Primary structure Enzyme location Signal sequence

Structura primara Locatie enzima Secventa semnal

Caracterizarea enzimei

Enzyme characterization

Determinarea structurii primare Determination of primary structure

Primary structure Enzyme location Signal sequence

www1.lf1.cuni.cz

Structura primara

Locatie proteina

Secventa semnal / Signal sequence

https://www.uniprot.org/blast/

Reactii catalizate de enzime din E. coli

otsA - trehaloz fosfat sintaza (TPS)

otsB - trehaloz fosfat fosfataza (TPP)

treC - trehaloz fosfat hidrolaza (TPH)

treF - trehalaza

Schluepmann, H. & all (2003),

PNAS,100, 6849-6854

Caracterizarea enzimei

Enzyme characterization

Primary structure

Enzyme location

http://www.cbs.dtu.dk/services/SignalP/

Caracterizarea enzimei

Enzyme characterization

Determinarea structurii primare Determination of primary structure

Structura primara Locatie enzima Secventa semnal

Primary structure Enzyme location Signal sequence

Acil-CoA

dehidrogenaza

umană

PubMed AAA74424.1

Caracterizarea enzimei

Enzyme characterization

Determinarea structurii primare Determination of primary structure

Structura primara Locatie enzima

Primary structure Enzyme location Signal sequence

Human

Acyl-CoA

dehydrogenase

PubMed AAA74424.1

Phobius

Caracterizarea enzimei

Enzyme characterization

Determinarea structurii primare Determination of primary structure

Structura primara Locatie enzima

Primary structure Enzyme location Signal sequence

Secventa semnal Phobius

http://phobius.sbc.su.se/

Caracterizarea enzimei

Enzyme characterization

Determinarea structurii primare Determination of primary structure

Structura primara Locatie enzima

Primary structure Enzyme location

P0DJD7

Pepsina umana 1-388

Signal sequence

Caracterizarea enzimei

Enzyme characterization

Determinarea structurii primare Determination of primary structure

Structura primara Locatie enzima

Primary structure Enzyme location

Secventa semnal / Signal sequences Signal-3L 2.0 prediction

http://www.csbio.sjtu.edu.cn/bioinf/Signal-3L/

Rezultate via Email – lent

Results via Email-slow

Estimarea proteinelor din S. Cerevisiae - peptida semnal

Estimation of S. cerevisiae proteins – signal sequences

Yofe, I. et al., Nature Methods,

13, 371-378 (2016)

Caracterizarea enzimei

Enzyme characterization

Determinarea structurii primare Determination of primary structure

Structura primara Locatie enzima

Primary structure Enzyme location

MKWLLLLGLVALSECIMYKVPLIRKKSLRRTLSERGLLKDFLKKHNLNP

ARKYFPQWEAPTLVDEQPLENYLDMEYFGTIGIGTPAQDFTVVFDTGS

SNLWVPSVYCSSLACTNHNRFNPEDSSTYQSTSETVSITYGTGSMTG

ILGYDTVQVGGISDTNQIFGLSETEPGSFLYYAPFDGILGLAYPSISSSGA

TPVFDNIWNQGLVSQDLFSVYLSADDQSGSVVIFGGIDSSYYTGSLNW

VPVTVEGYWQITVDSITMNGEAIACAEGCQAIVDTGTSLLTGPTSPIANI

QSDIGASENSDGDMVVSCSAISSLPDIVFTINGVQYPVPPSAYILQSEG

SCISGFQGMNLPTESGELWILGDVFIRQYFTVFDRANNQVGLAPVA

6 C 3 punti disulfidice

4 W fluorescenta

Caracterizarea enzimei

Enzyme characterization

Determinarea structurii primare Determination of primary structure

Structura primara Localizare AA

Primary structure AA localization

6 C 3 disulfide bridges

4 W fluorescence

Similaritati cu alte peptide

din baze de date existente

Similarities to other peptides

in existing databases

http://www.uniprot.org/

peptidesearch/

Caracterizarea enzimei

Enzyme characterization

Determinarea structurii primare Determination of primary structure

Structura primara Grad de similitudine enzime

Primary structure Comparisons between enzymes

Cautare peptida Peptide search

Caracterizarea enzimei

Enzyme characterization

Determinarea structurii primare Determination of primary structure

Structura primara Grad de similitudine enzime

Primary structure Comparisons between enzymes

Clustal Omega https://www.ebi.ac.uk/Tools/msa/clustalo/

P0DJD7, C1HAC9

Se aleg secventele in format FASTA (UNIPROT)

Caracterizarea enzimei

Enzyme characterization

Determinarea structurii primare Determination of primary structure

Structura primara Grad de similitudine enzime

Primary structure Comparisons between enzymes

FASTA (UNIPROT) sequences are selected

Kalign https://www.ebi.ac.uk/Tools/msa/kalign/

Caracterizarea enzimei

Enzyme characterization

Determinarea structurii primare Determination of primary structure

Structura primara Grad de similitudine enzime

Primary structure Comparisons between enzymes

Caracterizarea enzimei

Enzyme characterization

Determinarea structurii primare Determination of primary structure

Structura primara Grad de similitudine enzime

Primary structure Comparisons between enzymes

CLUSTAL 2.1 http://www.ebi.ac.uk/Tools/msa/clustalw2/

P0DJD7 Q4R4P0 C1HAC9

Se aleg secventele in format FASTA (UNIPROT)

FASTA (UNIPROT) sequences are selected

Caracterizarea enzimei

Enzyme characterization

Determinarea structurii primare Determination of primary structure

Structura primara Grad de similitudine enzime

Primary structure Comparisons between enzymes

Distanța evolutivă (arborele filoproteic)

Similarity tree (phylogram)

mitogen-activated protein kinase 14 (MAPKK)

atlasgeneticsoncology.org/Genes/

Caracterizarea enzimei

Enzyme characterization

Determinarea structurii primare Determination of primary structure

Distanța evolutivă (arborele filoproteic)

Similarity tree (phylogram)

PIR (Protein informatic resource)

http://pir.georgetown.edu/pirwww/search/pairwise.shtml

P0DJD7, C1HAC9

Se aleg secventele in format FASTA / codul de acces (UNIPROT)

FASTA (UNIPROT) sequence / access code is selected

Caracterizarea enzimei

Enzyme characterization

Determinarea structurii primare Determination of primary structure

Structura primara Grad de similitudine enzime

Primary structure Comparisons between enzymes

Pfam Colectie baza de date

familii de proteine

http://pfam.xfam.org/

Aspartil proteaza

Aspartyl protease

Caracterizarea enzimei

Enzyme characterization

Determinarea structurii primare Determination of primary structure

Structura primara Tipul/Clasa?

Primary structure Type/Class ?

Pfam Database collection

of protein families

http://pfam.xfam.org/

Cu/Zn superoxid dismutaza

Alvaro G. Estévez, Ed., Mental and Behavioural

Disorders and Diseases of the Nervous System »

"Current Advances in Amyotrophic Lateral Sclerosis„

(2013), Chapter 5.

Pfam

Caracterizarea enzimei

Enzyme characterization

Determinarea structurii primare Determination of primary structure

Structura primara Tipul/Clasa?

Primary structure Type/Class ?

PhosphoELM

Caracterizarea enzimei

Enzyme characterization

Determinarea structurii primare Determination of primary structure

Structura primara situsuri de fosforilare

Primary structure phosphorylation site

prediction

PhosphoELM phospho.elm.eu.org

Caracterizarea enzimei

Enzyme characterization

Determinarea structurii primare Determination of primary structure

Structura primara situsuri de fosforilare

Primary structure phosphorylation site

prediction