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

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Hydrolytic enzymes

Hydrolytic enzymes

Zn(II) containing enzymes


Hydrolytic enzymes

Enzymaticcatalysis of hydrolysis


Hydrolytic enzymes

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.


Hydrolytic enzymes

Carboanhydrase (CA)

  • Human carboanhydrase II

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


  • Hydrolytic enzymes

    Carboanhydrase

    pK = 6.8


    Hydrolytic enzymes

    Carboanhydrase

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


    Hydrolytic enzymes

    Carboanhydrase

    • The role of the metal ion:

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

    • Electrostatic stabilisation of the transient state


    Hydrolytic enzymes

    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.


    Hydrolytic enzymes

    Hydolysis of phosphoricacidesters

    The role of the metal ions:

    Inthecase of multimetalcentres, the metal ionsmaycooperateincompletingthetaskormaydevidethedutiesbetweenthem.


    Hydrolytic enzymes

    Alkalinephosphatase


    Hydrolytic enzymes

    Alkalinephosphatase

    The „ping-pong” mechanism


    Hydrolytic enzymes

    Purpleacidphosphatase


    Hydrolytic enzymes

    Purpleacidphosphatase


    Hydrolytic enzymes

    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.


    Hydrolytic enzymes

    Amino acid sequence of the purple acid phosphatases from various organisms


    Hydrolytic enzymes

    Phosphoricaciddiesterases

    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.


    Hydrolytic enzymes

    Phosphoricaciddiesterases

    The schematic structure of the active centre of the staphylococcus nuclease


    Hydrolytic enzymes

    Restrictionendonucleases


    Hydrolytic enzymes

    Restrictionendonucleases

    The complex of EcoRI restriction endonuclease formed with DNA


    Hydrolytic enzymes

    Restrictionendonucleases

    The complex of BamHI restriction endonuclease formed with DNA


    Hydrolytic enzymes

    Restrictionendonucleases

    The EcoRV restriction endonuclease


    Hydrolytic enzymes

    Restrictionendonucleases

    Structure of the active centre of EcoRV restriction endonuclease enzyme


    Hydrolytic enzymes

    Restrictionendonucleases

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


    Hydrolytic enzymes

    Restrictionendonucleases

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


    Hydrolytic enzymes

    Artificial zinc finger nucleases

    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.


    Hydrolytic enzymes

    The zinc finger motif

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


    Hydrolytic enzymes

    Alfred Pingoud, George H Silva:

    Precision genome surgery

    NATURE BIOTECHNOLOGY,

    2007, 25(7), 743-744


    Hydrolytic enzymes

    HNH-nucleases

    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.


    Hydrolytic enzymes

    HNH-nucleases


    Hydrolytic enzymes

    A colicinek

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


    Hydrolytic enzymes

    HNH-nucleases

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


    Hydrolytic enzymes

    Proteases, peptidases

    Hydrophobic

    pocket

    Active centre of carboxypeptidase A


    Hydrolytic enzymes

    Proteases, peptidases

    Hydrophobic

    pocket

    Active centre of carboxypeptidase A and mechanism of the reaction


    Hydrolytic enzymes

    Endopeptidases

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


    Hydrolytic enzymes

    Endopeptidases

    BaP1 metalloproteinase


    Hydrolytic enzymes

    Endopeptidases

    Human MMP12


    Hydrolytic enzymes

    The urease

    Non catalysed reaction:

    Catalysed reaction:


    Hydrolytic enzymes

    The urease

    Mechanism of the urease enzyme


    Hydrolytic enzymes

    β-lactamase

    Substrates:


    Hydrolytic enzymes

    β-lactamase

    Mechanism of β-lactamase enzyme


    Hydrolytic enzymes

    Ribozymes

    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).


    Hydrolytic enzymes

    Ribozymes

    Reaction mechanism of the action of large ribozymes

    BOH = H2O (RNase P),

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


    Hydrolytic enzymes

    Ribozymes

    Reaction mechanism of the reactions catalysed by the smaller ribozymes


    Hydrolytic enzymes

    Ribozymes

    Hydrolysis of pre-tRNSAsp catalysed by Rnase P


    Hydrolytic enzymes

    Ribozymes

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


    Hydrolytic enzymes

    Ribozymes

    • 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.


    Hydrolytic enzymes

    Alcohol-dehydrogenase enzymes


    Hydrolytic enzymes

    Alcohol-dehydrogenase enzymes

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


    Hydrolytic enzymes

    Alcohol-dehydrogenase enzymes

    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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