Caracterizarea enzimei - · PDF file subtilisina digestia Cistein proteaze situsul activ...

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  • 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