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Hydrolytic enzymes

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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  1. Hydrolytic enzymes Zn(II) containing enzymes

  2. Enzymaticcatalysis of hydrolysis

  3. 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.

  4. Carboanhydrase (CA) • Human carboanhydrase II • Rate is higher by 7-8 orders of magnitude  diffusion controlled limit

  5. Carboanhydrase pK = 6.8

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

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

  8. 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.

  9. Hydolysis of phosphoricacidesters The role of the metal ions: Inthecase of multimetalcentres, the metal ionsmaycooperateincompletingthetaskormaydevidethedutiesbetweenthem.

  10. Alkalinephosphatase

  11. Alkalinephosphatase The „ping-pong” mechanism

  12. Purpleacidphosphatase

  13. Purpleacidphosphatase

  14. 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.

  15. Amino acid sequence of the purple acid phosphatases from various organisms

  16. 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.

  17. Phosphoricaciddiesterases The schematic structure of the active centre of the staphylococcus nuclease

  18. Restrictionendonucleases

  19. Restrictionendonucleases The complex of EcoRI restriction endonuclease formed with DNA

  20. Restrictionendonucleases The complex of BamHI restriction endonuclease formed with DNA

  21. Restrictionendonucleases The EcoRV restriction endonuclease

  22. Restrictionendonucleases Structure of the active centre of EcoRV restriction endonuclease enzyme

  23. Restrictionendonucleases Structure of the Ca2+ binding site of the EcoRV restriction endonuclease enzyme

  24. Restrictionendonucleases Dimerisation of the nuclease domen of the FokI restriction endonuclease on the substrate molecule

  25. 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.

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

  27. Alfred Pingoud, George H Silva: Precision genome surgery NATURE BIOTECHNOLOGY, 2007, 25(7), 743-744

  28. 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.

  29. HNH-nucleases

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

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

  32. Proteases, peptidases Hydrophobic pocket Active centre of carboxypeptidase A

  33. Proteases, peptidases Hydrophobic pocket Active centre of carboxypeptidase A and mechanism of the reaction

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

  35. Endopeptidases BaP1 metalloproteinase

  36. Endopeptidases Human MMP12

  37. The urease Non catalysed reaction: Catalysed reaction:

  38. The urease Mechanism of the urease enzyme

  39. β-lactamase Substrates:

  40. β-lactamase Mechanism of β-lactamase enzyme

  41. 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).

  42. Ribozymes Reaction mechanism of the action of large ribozymes BOH = H2O (RNase P), BOH = 2’-hydroxyl group of guanosin cofactor (type I intron)

  43. Ribozymes Reaction mechanism of the reactions catalysed by the smaller ribozymes

  44. Ribozymes Hydrolysis of pre-tRNSAsp catalysed by Rnase P

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

  46. 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.

  47. Alcohol-dehydrogenase enzymes

  48. Alcohol-dehydrogenase enzymes Structure and NADH binding site of the ADH enzyme of Pseudomonas aeruginosa

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