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

Impachetare 3D 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

Edman Edman

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 cleavage Proteaza Protease Natura legaturii peptidice clivate

Peptide bond selectivity

N-terminal -----XYZ----------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/

https://www.google.com/url?sa=i&rct=j&q=&esrc=s&source=images&cd=&cad=rja&uact=8&ved=2ahUKEwiB3Nj8gIrhAhXEbFAKHV15D2cQjRx6BAgBEAU&url=https%3A%2F%2Fwww.mpi-magdeburg.mpg.de%2F47582%2FSoftware_Development_for_Multiplexing_CGE-LIF_Based_HT_HP_HR_Glycoanalysis&psig=AOvVaw0SshcAfL3_7WyHwz2n5384&ust=1552940662146720

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

pura Reactii de

clivare

Etapa 1 Amestec de

fragmente Separarea

fragmentelor

Etapa 2 Fragmente

purificate

Etapa 3

secventierea

Set de

secvente

individuale

Etapa 4

-clivarea

alternativa;

-separarea si

secventierea Secventa

completa

Amestec

complex Proteoliza

Peptide Separarea

fragmentelor

HPLC Fragmente

purificate

LC-MS/MS

Izolare

peptida Fragmentare

(Spectru de masa)

Tripsina

Baza de date

Secvente

Complete

Caracterizarea enzimei

Enzyme characterization

Determinarea structurii primare Determination of primary structure

Pure

Enzyme Cleavage

reaction

Step 1 Mixture of

fragments Fragments

separation

Step 2 Purified

fragments

Step 3

sequencing

Set of

individual

sequences

Step 4

-alternative

cleavage;

-separation and

sequencing Full

sequence

Complex

mixture Proteolysis

Peptides Separation

HPLC Purified

fragments

LC-MS/MS

Peptide

Isolation Fragmentation

(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 A2 ALWQF

interacțiuni cu

substratul

nepolar Stabilirea

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 A2 ALWQF

interactions with

nonpolar

substrate To 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