Hydrolytic enzymes ZnII containing enzymes Enzymatic catalysis of

Hydrolytic enzymes Zn(II) containing enzymes

Enzymatic catalysis of hydrolysis Enzyme Metal ion(s) Catalyzed reaction Alkaline phosphatase Purple acid phosphatase Phosphoprotein phosphatase 2 Zn. II, 1 Mg 2+ 1 Fe. III, 1 Zn. II Staphylococcal nuclease DNA polymerase 1 Ribonuclease H Phospholipase A Thermolysin Carboxypeptidase A Adamalysin Urease b-lactamase Arginase 1 Ca 2+ 2 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 b-lactam ring Hydrolysis of guanidium group of arginine

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.

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

Carboanhydrase p. K = 6. 8

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

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

Hydolysis of phosphoric acid esters SN 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.

Hydolysis of phosphoric acid esters 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.

Alkaline phosphatase

Alkaline phosphatase The „ping-pong” mechanism

Purple acid phosphatase

Purple acid phosphatase

Purple acid phosphatase The strong Lewis acid Fe. III ion is responsible for generating the nucleophile OH(this is the reason for the acidic p. H-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.

Amino acid sequence of the purple acid phosphatases from various organisms

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 Mn. A in the mechanism of the enzymatic reaction.

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

Restriction endonucleases

Restriction endonucleases The complex of Eco. RI restriction endonuclease formed with DNA

Restriction endonucleases The complex of Bam. HI restriction endonuclease formed with DNA

Restriction endonucleases The Eco. RV restriction endonuclease

Restriction endonucleases Structure of the active centre of Eco. RV restriction endonuclease enzyme

Restriction endonucleases Structure of the Ca 2+ binding site of the Eco. RV restriction endonuclease enzyme

Restriction endonucleases Dimerisation of the nuclease domen of the Fok. I restriction endonuclease on the substrate molecule

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 Fok. I restriction endonuclease.

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

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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.

HNH-nucleases

A colicinek A Colicin E 7 HNH-nukleáz és a DNS molekula komplexe.

HNH-nucleases A Colicin E 7 HNH-nukleáz domén C-, és N-terminális részének együttműködése: az N-terminális arginin szükséges a katalitikus aktivitáshoz – allosztérikus kontroll.

Proteases, peptidases Hydrophobic pocket Active centre of carboxypeptidase A

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

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

Endopeptidases Ba. P 1 metalloproteinase

Endopeptidases Human MMP 12

The urease Non catalysed reaction: Catalysed reaction:

The urease Mechanism of the urease enzyme

β-lactamase Substrates:

β-lactamase Mechanism of β-lactamase enzyme

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

Ribozymes Reaction mechanism of the reactions catalysed by the smaller ribozymes

Ribozymes Hydrolysis of pre-t. RNSAsp catalysed by Rnase P

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

Ribozymes 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.

Alcohol-dehydrogenase enzymes

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

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.