hydrolytic enzymes zn(ii) containing enzymes. enzymatic catalysis of hydrolysis enzymemetal...
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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
