Hydrolytic enzymes
Zn(II) containing enzymes
Enzymatic catalysis of hydrolysis
Enzyme
Metal ion(s)
Catalyzed reaction
Alkaline phosphatase
Purple acid phosphatase
Phosphoprotein phosphatase
2 ZnII, 1 Mg2+
1 FeIII, 1 ZnII
1 FeIII, 1 ZnII
Staphylococcal nuclease
DNA polymerase 1
Ribonuclease H
Phospholipase A
Thermolysin
Carboxypeptidase A
Adamalysin
Urease
b-lactamase
Arginase
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 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
Leu-196
Leu-196
H
H
O
Thr-199
Thr-199
Val-143
H
H
Val-121
- HCO 3-
H
O-
- H+
Val-121
Zn
His
His
His
Val-143
pK = 6.8
O
Zn
O
His
His
His
1
4
+ CO 2
2
+ H 2O
Leu-196
Leu-196
H
H
Thr-199
O
O
H
Thr-199
Val-143
C
O
O
H
O
O
C
O
O
Val-121
Val-121
Zn
His
His
His
Val-143
3
Zn
His
His
His
Carboanhydrase
O
C
Glu-106
OTyr-7
Leu-196
H
Thr-199
OH
O
Val-143
H
HN
N
H2O
O
H2O
H
Zn
His
His
His
Val-121
O
C
His-64
O
Asn-244
O
C
Glu-117
O
C
Gln-92
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
SN2 mechanism:
R
O
O
- H+
P
R
P
O
O
O
O
OH2
O
O
O
++
M
M
R
P
O
+ H+
O
O
-
++
++
R
O
O
R
R
R
R
-
M
HO
O
P
OH
OH
M++
OH
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
W
W
Asp-327
His-331 Zn1
His-412
His-370
W
W
Zn1
W
H2N
W
Arg-166
H2N
O
Asp-369 Zn2 H
O
Asp-51 C
OH
O
W Mg W
Glu-322
H2N
H2N
O
Zn2
Ser-102
+ ROPO32
- 2H2O
Ser-102
O
C
OH2
O
Mg
H
O H
Zn1 R
O
O
H2N
P
O
H2N
O
Zn2
O
O
Ser-102
C
OH
2
O
Mg
Thr-155
OH
- HPO42
+ 3H2O
Zn1
H
O
P
O
Zn2
O
H
O
Mg
Zn1
H2N
O
H2N
Zn2
O Ser-102
C
O
O
O
H2N
P O
H2N
O
O
H
C
Ser-102
O
O
H
O
Mg
H
The „ping-pong” mechanism
H2O
E
Zn
E•ROP
H2O
1
H2O
Zn
R
1
O
H2N
Zn
2
O
O
Zn
CH2
H
Asp(51)
C O-
NH
Arg(166)
H2N
+ R-OP
Ser(102)
-
OH
Mg
O
3
O
Asp(51)
C O-
H2O
Glu(322)
2-
-PO4
2
O
O
-
O
O
OH2
O
Glu(322)
-
3
O
-
HO
H2O
CH2 CH3
-
+H2O
O
O
H2N
-RO-
O
H2N
-
OH
+ C
O
NH
Arg(166)
Zn
1
O
-
Zn
CH2
Ser(102)
HO
H2O
O
OH2
O
H2N
O
H2N
+ C
O
P
2
O
NH
Arg(166)
-
CH2
Ser(102)
O
H2O
CH2 CH3
Thr(155)
C
3
C
Asp(51)
C O-
Mg
NH
Arg(166)
Thr(155)
2-
O
H2O
+ C
O
1
P
Asp(51)
C O-
Mg
O
O
Zn
H2N
CH2
Ser(102)
OH2
Thr(155)
Glu(322)
C
Zn
O
O
CH2 CH3
+PO4
H2N
-
O
HO
H2O
-
O
2
O
H2O
O
P
+ C
O
Mg
O
E•Pi
Glu(322)
-
3
HO
H2O
CH2 CH3
Thr(155)
C
O
E-P
Purple acid phosphatase
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.
Amino acid sequence of the purple acid phosphatases from
various organisms
Mammals:
Uf
bsPAP
human PAP
rat PAP
mouse PAP
10
9
10
9
9
VAVGDWGGVPN
VAVGDWGGVPN
VAVGDWGGVPN
VAVGDWGGVPN
VAVGDWGGVPN
26
26
26
26
26
ILSLGDNFYFTG
VXSXGDNFYFSX
ILSLGDNFYFTG
IMSLGDNFYFTG
IMSLGDNFYFTG
28
28
28
28
28
VLAGNHDHLG
XXAGNHDHXG
VLAGNHDHLG
VLAGNHDHLG
VLAGNHDHLG
84
74
84
86
86
VAGHYPVW
VAGHYPVW
VAGHYPVW
VAGHYPIW
VAGHYPIW
26
26
26
26
26
YLCGHDHNLQ
YXCGHDHNXQ
YLCGHDHNLQ
YLCGHDHNLQ
YLCGHDHNLQ
Plants:
kbPAP
atPAP
131 GLIGDLGQSFD 17 VLFVGDLSYADR 26 WTAGNHEIEF
164 GLIGDLGQTYD 17 VLFVGDLSYADR 26 WTAGNHEIDF
Bacteria:
afPAP
164 AVLNDMGYTNA 17 AWHGGDLSYADD 78 VLPGNHEASC 147 VMSHRPMY 26 YLSGHIHWYE
76 VLMHSPLY 28 VFAGHVHAYE
76 VLVHSPFY 28 VFAGHVHAYE
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.
Phosphoric acid diesterases
Arg-87
R
O
O
P
Arg-35
Asp-40
Val-41
O
Ca
R
O
O
H
O
OH2
Asp-21
H
H
H O
O
C
Glu-43
The schematic structure of the active centre of the staphylococcus
nuclease
Restriction endonucleases
Arg Asp End
CGT GAT TGA
Restriction endonucleases
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
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
HNH-nucleases
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
A colicinek
A Colicin E7 HNH-nukleáz és a DNS
molekula komplexe.
HNH-nucleases
A Colicin E7 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
BaP1 metalloproteinase
Endopeptidases
Human MMP12
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 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)
Ribozymes
Reaction mechanism of the reactions catalysed by the smaller ribozymes
Ribozymes
Hydrolysis of pre-tRNSAsp catalysed by Rnase P
Ribozymes
Secondary and tertiary structures of the RNA of the RNase P of E. coli.
12.17.ábra
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.