1. Hydrolytic enzymes Zn(II) containing enzymes

2. Enzymatic catalysis of hydrolysis EnzymeMetal ion(s)Catalyzed reaction Alkaline phosphatase Purple acid phosphatase Phosphoprotein phosphatase Staphylococcal nuclease DNA polymerase 1 Ribonuclease H Phospholipase A Thermolysin Carboxypeptidase A Adamalysin Urease  -lactamase Arginase 2 Zn II, 1 Mg 2+ 1 Fe III, 1 Zn II 1 Ca 2+ 2 Mg 2+ 1 Mg 2+ 1 Ca 2+ 1 Zn II 2 Ni II 2 Zn II 2 Mn II Hydrolysis of Phosphoric acid monoester Hydrolysis of phosphoricester bond of phosphoproteins Hydrolysis of DNA Hydrolysis of RNA Hydrolysis of phospholipids Hydrolysis of intrachain peptid bond in proteins Hydrolysis peptide bond of C-terminal residues Hydrolysis of peptide bond in proteins Hydrolyisis of carbamide Hydrolyisis of  -lactam ring Hydrolysis of guanidium group of arginine

3. Hydrolytic enzymes 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. The hydrogen bond network in the active centre of human carboanhydrase. Carboanhydrase

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

8. Hydolysis of phosphoric acid esters S N 2 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. The role of the metal ions: In the case of multimetal centres, the metal ions may cooperate in completing the task or may devide the duties between them. Hydolysis of phosphoric acid esters

10. Alkaline phosphatase

11. The „ping-pong” mechanism Alkaline phosphatase

13. Purple acid phosphatase

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

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

17. Phosphoric acid diesterases 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.

18. The schematic structure of the active centre of the staphylococcus nuclease Phosphoric acid diesterases

19. Restriction endonucleases

21. The complex of EcoRI restriction endonuclease formed with DNA Restriction endonucleases

22. The complex of BamHI restriction endonuclease formed with DNA Restriction endonucleases

23. The EcoRV restriction endonuclease Restriction endonucleases

24. Structure of the active centre of EcoRV restriction endonuclease enzyme Restriction endonucleases

25. Structure of the Ca 2+ binding site of the EcoRV restriction endonuclease enzyme Restriction endonucleases

26. Dimerisation of the nuclease domen of the FokI restriction endonuclease on the substrate molecule Restriction endonucleases

27. 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.

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

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

30. 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

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

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

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

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

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

37. BaP1 metalloproteinase Endopeptidases

38. Human MMP12 Endopeptidases

39. The urease Non catalysed reaction: Catalysed reaction:

40. Mechanism of the urease enzyme The urease

41. β-lactamase Substrates:

42. Mechanism of β-lactamase enzyme β-lactamase

43. 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).

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

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

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

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

48. The transient state of the hydrolytic process catalysed by the ribozyme of RNase P of E coli. The metal ion may function as: (i)Formation of the tertiary structure of the RNA, (ii) Binding the substrate, and/or (iii) Participate in the catalytic cycle.

49. Alcohol-dehydrogenase enzymes

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

51. 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. Alcohol-dehydrogenase enzymes