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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

slide3

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.
slide4

Carboanhydrase (CA)

        • Human carboanhydrase II
  • Rate is higher by 7-8 orders of magnitude  diffusion controlled limit
slide6

Carboanhydrase

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

slide7

Carboanhydrase

  • The role of the metal ion:
  • a nucleophile reactant, i.e. formation of a hydroxide ion
  • Electrostatic stabilisation of the transient state
slide8

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.

slide9

Hydolysis of phosphoricacidesters

The role of the metal ions:

Inthecase of multimetalcentres, the metal ionsmaycooperateincompletingthetaskormaydevidethedutiesbetweenthem.

slide11

Alkalinephosphatase

The „ping-pong” mechanism

slide15

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.

slide17

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.

slide18

Phosphoricaciddiesterases

The schematic structure of the active centre of the staphylococcus nuclease

slide21

Restrictionendonucleases

The complex of EcoRI restriction endonuclease formed with DNA

slide22

Restrictionendonucleases

The complex of BamHI restriction endonuclease formed with DNA

slide23

Restrictionendonucleases

The EcoRV restriction endonuclease

slide24

Restrictionendonucleases

Structure of the active centre of EcoRV restriction endonuclease enzyme

slide25

Restrictionendonucleases

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

slide26

Restrictionendonucleases

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

slide27

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.

slide28

The zinc finger motif

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

slide29

Alfred Pingoud, George H Silva:

Precision genome surgery

NATURE BIOTECHNOLOGY,

2007, 25(7), 743-744

slide30

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.

slide32

A colicinek

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

slide33

HNH-nucleases

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

slide34

Proteases, peptidases

Hydrophobic

pocket

Active centre of carboxypeptidase A

slide35

Proteases, peptidases

Hydrophobic

pocket

Active centre of carboxypeptidase A and mechanism of the reaction

slide36

Endopeptidases

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

slide37

Endopeptidases

BaP1 metalloproteinase

slide38

Endopeptidases

Human MMP12

slide39

The urease

Non catalysed reaction:

Catalysed reaction:

slide40

The urease

Mechanism of the urease enzyme

slide41

β-lactamase

Substrates:

slide42

β-lactamase

Mechanism of β-lactamase enzyme

slide43

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).

slide44

Ribozymes

Reaction mechanism of the action of large ribozymes

BOH = H2O (RNase P),

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

slide45

Ribozymes

Reaction mechanism of the reactions catalysed by the smaller ribozymes

slide46

Ribozymes

Hydrolysis of pre-tRNSAsp catalysed by Rnase P

slide47

Ribozymes

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

slide48

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.
slide50

Alcohol-dehydrogenase enzymes

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

slide51

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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