Hydrolytic enzymes
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Hydrolytic enzymes. Zn(II) containing enzymes. Enzymatic catalysis of hydrolysis. Hydrolytic enzymes. Characteristics of the zinc(II) ion: redoxi inert, strong Lewis acid, forms strong coordinative bonds,

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Hydrolytic enzymes

Hydrolytic enzymes

Zn(II) containing enzymes


Enzymaticcatalysis of hydrolysis


Hydrolyticenzymes

  • Characteristics of the zinc(II) ion:

  • redoxi inert,

  • strong Lewis acid,

  • forms strong coordinative bonds,

  • Because of the saturated d shell, the crystal field stabilisation is zero, and thus the coordination number and geometry easily change in its complexes.


Carboanhydrase (CA)

  • Human carboanhydrase II

  • Rate is higher by 7-8 orders of magnitude  diffusion controlled limit


  • Carboanhydrase

    pK = 6.8


    Carboanhydrase

    The hydrogen bond network in the active centre of human carboanhydrase.


    Carboanhydrase

    • The role of the metal ion:

    • a nucleophile reactant, i.e. formation of a hydroxide ion

    • Electrostatic stabilisation of the transient state


    Hydolysis of phosphoricacidesters

    SN2 mechanism:

    Role of the metal ion:

    - Electrostatic activation of the substrate by coordination (Lewis acid activation), which will polarise the P–O bond, increasing the partial positive charge on the P atom, making the nuclephil attack easier,

    - Formation of the nucleophile reactant (mostly hydroxid ion).

    - Stabilisation of the phosphorane intermediate compound through charge compensation.

    - Stabilisation of the leaving group by coordination.


    Hydolysis of phosphoricacidesters

    The role of the metal ions:

    Inthecase of multimetalcentres, the metal ionsmaycooperateincompletingthetaskormaydevidethedutiesbetweenthem.


    Alkalinephosphatase


    Alkalinephosphatase

    The „ping-pong” mechanism


    Purpleacidphosphatase


    Purpleacidphosphatase


    Purpleacidphosphatase

    The strong Lewis acid FeIII ion is responsible for generating the nucleophile OH- (this is the reason for the acidic pH-optimum), while the ZnII ion is responsible for binding and activating electrostatically the substrate. In the stabilisation of the phosphoran intermediate compound both metal ions participate.



    Phosphoric various organismsaciddiesterases

    The active centre of the Klenow-fragment 3’-5’-exonuclease subunit, the way of binding the substrate, and the role of the hidoxide ion bound to MnA in the mechanism of the enzymatic reaction.


    Phosphoric various organismsaciddiesterases

    The schematic structure of the active centre of the staphylococcus nuclease


    Restriction various organismsendonucleases


    Restriction various organismsendonucleases

    The complex of EcoRI restriction endonuclease formed with DNA


    Restriction various organismsendonucleases

    The complex of BamHI restriction endonuclease formed with DNA


    Restriction various organismsendonucleases

    The EcoRV restriction endonuclease


    Restriction various organismsendonucleases

    Structure of the active centre of EcoRV restriction endonuclease enzyme


    Restriction various organismsendonucleases

    Structure of the Ca2+ binding site of the EcoRV restriction endonuclease enzyme


    Restriction various organismsendonucleases

    Dimerisation of the nuclease domen of the FokI restriction endonuclease on the substrate molecule


    Artificial zinc finger nucleases various organisms

    The artificial zinc finger nucleases are coupled proteins in which the specific DNA binding is provided by the zinc fingers, while cleavage of DNA is made by a nuclease domen – usually the cleaving domen of the FokI restriction endonuclease.


    The zinc finger motif various organisms

    The structure of the zinc finger motif is formed by coordination of the zinc(II) ion.


    Alfred Pingoud various organisms, George H Silva:

    Precision genome surgery

    NATURE BIOTECHNOLOGY,

    2007, 25(7), 743-744


    HNH-nucleases various organisms

    A HNH-motívum szerkezete a cink-ujj szerkezethez hasonló, de a cinkion koordinációja más. Itt a fémion három hisztidin oldallánchoz kapcsolódik, és a szabadon maradt koordinációs helyet egy, a DNS foszfátészter kötéséből származó oxigén donoratom foglalja el. Ebből adódóan a funkció is megváltozott: DNS szabályozás helyett DNS hasítás.


    HNH-nucleases various organisms


    A various organismscolicinek

    A Colicin E7 HNH-nukleáz és a DNS molekula komplexe.


    HNH-nucleases various organisms

    A Colicin E7 HNH-nukleáz domén C-, és N-terminális részének együttműködése: az N-terminális arginin szükséges a katalitikus aktivitáshoz – allosztérikus kontroll.


    Proteases various organisms, peptidases

    Hydrophobic

    pocket

    Active centre of carboxypeptidase A


    Proteases various organisms, peptidases

    Hydrophobic

    pocket

    Active centre of carboxypeptidase A and mechanism of the reaction


    Endopeptidases various organisms

    Active centre of thermolysin (a) and adamalysin II (b) enzymes


    Endopeptidases various organisms

    BaP1 metalloproteinase


    Endopeptidases various organisms

    Human MMP12


    The various organismsurease

    Non catalysed reaction:

    Catalysed reaction:


    The various organismsurease

    Mechanism of the urease enzyme


    β-lactamase various organisms

    Substrates:


    β-lactamase various organisms

    Mechanism of β-lactamase enzyme


    Ribozymes various organisms

    Characteristics of RNA:

    (i) The four possible side chains (base) as compared with the proteins provide significantly less structural variety,

    (ii) The bases are not able the uptake or liberation of protons in the physiological pH range (catalysis of acid-base processes is not favoured),

    (iii) the RNA chain is fairly flexible (precise positionation of the substrate is difficult), and

    (iv) It has high negative charge (the possibility of nonspecific interactions with the charged substrates).


    Ribozymes various organisms

    Reaction mechanism of the action of large ribozymes

    BOH = H2O (RNase P),

    BOH = 2’-hydroxyl group of guanosin cofactor (type I intron)


    Ribozymes various organisms

    Reaction mechanism of the reactions catalysed by the smaller ribozymes


    Ribozymes various organisms

    Hydrolysis of pre-tRNSAsp catalysed by Rnase P


    Ribozymes various organisms

    Secondary and tertiary structures of the RNA of the RNase P of E. coli.


    Ribozymes various organisms

    • The transient state of the

    • hydrolytic process catalysed by

    • the ribozyme of RNase P of E coli.

    • The metal ion may function as:

    • Formation of the tertiary structure of

    • the RNA,

    • (ii) Binding the substrate, and/or

    • (iii) Participate in the catalytic cycle.



    Alcohol-dehydrogenase enzymes various organisms

    Structure and NADH binding site of the ADH enzyme of Pseudomonas aeruginosa


    Alcohol-dehydrogenase enzymes various organisms

    Active centre (the substrate analogue ethyleneglycole is bound to the zinc(II) ion) of the ADH enzyme of Pseudomonas aeruginosa. Protein Science (2004), 13:1547–1556.


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