Hydrolytic enzymes

Zn(II) containing enzymes

Enzymatic catalysis of hydrolysis

Enzyme Metal 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 ZnII, 1 Mg2+

1 FeIII, 1 ZnII

1 FeIII, 1 ZnII

1 Ca2+

2 Mg2+

1 Mg2+

1 Ca2+

1 ZnII

1 ZnII

1 ZnII

2 NiII

2 ZnII

2 MnII

Hydrolysis of Phosphoric acid monoester

Hydrolysis of Phosphoric acid monoester

Hydrolysis of phosphoricester bond of

phosphoproteins

Hydrolysis of DNA

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

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

O

HisHis

His

Zn

H H

O

H

Thr-199Val-143

Val-121

Leu-196 Leu-196

Val-121

Val-143Thr-199

O

HisHis

His

Zn

H

H

O

Leu-196

Val-121

Val-143Thr-199

C

O

OO

HisHis

His

Zn

H

H

O

Leu-196

Val-121

Val-143Thr-199 O

H

O

HisHis

His

Zn

H O

O

C

-

-

+ CO2

- H+

1

2

3

4- HCO3-

+ H2O

pK = 6.8

Leu-196

Val-121

Val-143

Thr-199 O

H

CO-

O

OH

NHN H2O H2O O

HisHis

His

ZnH

H

CO

O

C

OC

O

-

Asn-244

Gln-92

Glu-117

Glu-106

His-64

Tyr-7

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

Carboanhydrase

Hydolysis of phosphoric acid esters

OH2

M++

P

OO

O

OR

R

OH-M++

P

OO

O

OR

R

OH-

M++

PO

O O

OR

R

OH

POO O

R

M++

HOR

+ H+- H+

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.

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

Alkaline phosphatase

Asp-51 CO

O

Zn2

Mg

OH

W

Thr-155

W

Glu-322

His-370

Asp-369

O

HSer-102

W

WW

Zn1

Asp-327

His-331

His-412H2N

H2NArg-166

CO

O

Zn2

Mg

OH2

OSer-102

W

WW

Zn1H2N

H2NO

OP

O

O

RH2N

H2N

Zn1

CO

O

Zn2

Mg

OH2

OSer-102

O

HH

O P

O

O

H2N

H2N

CO

O

Zn2

Mg

O

O

Ser-102

OH

Zn1

H

H

+ ROPO32

Zn1

OP

O

O

H2N

H2N

CO

O

Zn2

MgO

H

H

OH

- 2H2O

- HPO42

+ 3H2O

O Ser-102

The „ping-pong” mechanism

Alkaline phosphatase

Zn1

H2O

H2O

H2NC

H2N

H2O

NH

Zn2 O

H CH2O

C

O

Mg3

OH-

H2O HOH2O

CH2 CH3

Ser(102)

Arg(166)

Asp(51)

Thr(155)

O-

O-

C

O

Glu(322)

O+

E

+ R-OP

E•ROP

-RO-

+H2O-PO4

2-+PO42-

E•Pi E-P

Zn1

O

P

O-

CH3CH2

H2OHOH2O Mg

3

O

C

OCH2

O-

Zn2

O

O

R

NHH2N

CH2N

Ser(102)Asp(51)

Thr(155)

O-

O-

C

O

Glu(322)

OH2

+OArg(166)

Ser(102)Asp(51)

Thr(155)

O-

O-

C

O

Glu(322)

CH3CH2

H2OHOH2O

OH2

Mg3

O

C

O

OH-

O

O

Zn2

O-

CH2

PO

Zn1

NHH2N

CH2N

+OArg(166)

Zn1

O

P

O-

CH3CH2

H2OHOH2O Mg

3

O

C

OCH2

O-

Zn2

O

O

OH2

NHH2N

CH2N

Ser(102)Asp(51)

Thr(155)

O-

O-

C

O

Glu(322)

+OArg(166)

Purple acid phosphatase

Purple acid phosphatase

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.

Purple acid phosphatase

Amino acid sequence of the purple acid phosphatases from various organisms

Mammals:Uf 10 VAVGDWGGVPN 26 ILSLGDNFYFTG 28 VLAGNHDHLG 84 VAGHYPVW 26 YLCGHDHNLQ bsPAP 9 VAVGDWGGVPN 26 VXSXGDNFYFSX 28 XXAGNHDHXG 74 VAGHYPVW 26 YXCGHDHNXQ human PAP 10 VAVGDWGGVPN 26 ILSLGDNFYFTG 28 VLAGNHDHLG 84 VAGHYPVW 26 YLCGHDHNLQ rat PAP 9 VAVGDWGGVPN 26 IMSLGDNFYFTG 28 VLAGNHDHLG 86 VAGHYPIW 26 YLCGHDHNLQ mouse PAP 9 VAVGDWGGVPN 26 IMSLGDNFYFTG 28 VLAGNHDHLG 86 VAGHYPIW 26 YLCGHDHNLQ

Plants:kbPAP 131 GLIGDLGQSFD 17 VLFVGDLSYADR 26 WTAGNHEIEF 76 VLMHSPLY 28 VFAGHVHAYE atPAP 164 GLIGDLGQTYD 17 VLFVGDLSYADR 26 WTAGNHEIDF 76 VLVHSPFY 28 VFAGHVHAYE

Bacteria:afPAP 164 AVLNDMGYTNA 17 AWHGGDLSYADD 78 VLPGNHEASC 147 VMSHRPMY 26 YLSGHIHWYE

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.

The schematic structure of the active centre of the staphylococcus nuclease

CaOOH2

Asp-40Val-41

O

Asp-21

H

H

OC

O

Glu-43

PO

OO

RR

HO

H

Arg-87

Arg-35

Phosphoric acid diesterases

Restriction endonucleases

Arg Asp End CGT GAT TGA

The complex of EcoRI restriction endonuclease formed with DNA

Restriction endonucleases

The complex of BamHI restriction endonuclease formed with DNA

Restriction endonucleases

The EcoRV restriction endonuclease

Restriction endonucleases

Structure of the active centre of EcoRV restriction endonuclease enzyme

Restriction endonucleases

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

Restriction endonucleases

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

Restriction endonucleases

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.

The zinc finger motif

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

Alfred Pingoud, George H Silva: Precision genome surgery

NATURE BIOTECHNOLOGY, 2007, 25(7), 743-744

H-N-H endonuclease motif

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

HNH-nucleases

A colicinek

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

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

HNH-nucleases

Proteases, peptidases

Active centre of carboxypeptidase A

Hydrophobicpocket

Active centre of carboxypeptidase A and mechanism of the reaction

Hydrophobicpocket

Proteases, peptidases

Endopeptidases

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

BaP1 metalloproteinase

Endopeptidases

Human MMP12

Endopeptidases

The urease

Non catalysed reaction:

Catalysed reaction:

Mechanism of the urease enzyme

The urease

β-lactamase

Substrates:

Mechanism of β-lactamase enzyme

β-lactamase

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).

Ribozymes

Reaction mechanism of the action of large ribozymes

BOH = H2O (RNase P),

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

Reaction mechanism of the reactions catalysed by the smaller ribozymes

Ribozymes

Hydrolysis of pre-tRNSAsp catalysed by Rnase P

Ribozymes

12.17.ábraSecondary and tertiary structures of the RNA of the RNase P of E. coli.

Ribozymes

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 ofthe RNA,

(ii) Binding the substrate, and/or

(iii) Participate in the catalytic cycle.

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.

Alcohol-dehydrogenase enzymes