DB code: S00403

RLCP classification 1.15.9400.1180 : Hydrolysis
CATH domain 3.40.580.10 : ECO RI Endonuclease; Chain A Catalytic domain
E.C. 3.1.21.4
CSA
M-CSA
MACiE

CATH domain Related DB codes (homologues)

Uniprot Enzyme Name
UniprotKB Protein name Synonyms Pfam
P00642 Type-2 restriction enzyme EcoRI
R.EcoRI
EC 3.1.21.4
Type II restriction enzyme EcoRI
Endonuclease EcoRI
PF02963 (EcoRI)
[Graphical View]
P43642 Type-2 restriction enzyme MunI
R.MunI
EC 3.1.21.4
Type II restriction enzyme MunI
Endonuclease MunI
PF11407 (RestrictionMunI)
[Graphical View]

KEGG enzyme name
type II site-specific deoxyribonuclease
type II restriction enzyme

UniprotKB: Accession Number Entry name Activity Subunit Subcellular location Cofactor
P00642 T2E1_ECOLX Endonucleolytic cleavage of DNA to give specific double-stranded fragments with terminal 5''-phosphates. Homodimer. Binds 2 magnesium ions per subunit.
P43642 T2MU_MYCSP Endonucleolytic cleavage of DNA to give specific double-stranded fragments with terminal 5''-phosphates.

KEGG Pathways
Map code Pathways E.C.

Compound table
Cofactors Substrates Products Intermediates
KEGG-id C00305 C00039 C00001 C00578 C00039
E.C.
Compound Magnesium DNA H2O DNA 5'-phosphate DNA
Type divalent metal (Ca2+, Mg2+) nucleic acids H2O nucleic acids,phosphate group/phosphate ion nucleic acids
ChEBI 18420
15377
PubChem 888
22247451
962
1eriA Pdbj logo s Rasmollogo id Rasmollogo chain Mmcif id Mmcif chain Unbound Analogue:T-C-G-C-G-A-A-T-T-C-G-C-G (chain B) Unbound Unbound
1qc9A Pdbj logo s Rasmollogo id Rasmollogo chain Mmcif id Mmcif chain Unbound Unbound Unbound Unbound
1qc9B Pdbj logo s Rasmollogo id Rasmollogo chain Mmcif id Mmcif chain Unbound Unbound Unbound Unbound
1qc9C Pdbj logo s Rasmollogo id Rasmollogo chain Mmcif id Mmcif chain Unbound Unbound Unbound Unbound
1ckqA Pdbj logo s Rasmollogo id Rasmollogo chain Mmcif id Mmcif chain Unbound Bound:T-C-G-C-G-A-A-T-T-C-G-C-G (chain B) Unbound Unbound
1cl8A Pdbj logo s Rasmollogo id Rasmollogo chain Mmcif id Mmcif chain Unbound Bound:T-C-G-C-G-A-PRN-T-T-C-G-C-G (chain B) Unbound Unbound
1qpsA Pdbj logo s Rasmollogo id Rasmollogo chain Mmcif id Mmcif chain Analogue:_MN Unbound Bound:A-A-T-T-C-G-C-G (chain N) Bound:T-C-G-C-G (chain M)
1qrhA Pdbj logo s Rasmollogo id Rasmollogo chain Mmcif id Mmcif chain Unbound Bound:T-C-G-C-G-A-A-T-T-C-G-C-G (chain M) Unbound Unbound
1qriA Pdbj logo s Rasmollogo id Rasmollogo chain Mmcif id Mmcif chain Unbound Bound:T-C-G-C-G-A-A-T-T-C-G-C-G (chain M) Unbound Unbound
1d02A Pdbj logo s Rasmollogo id Rasmollogo chain Mmcif id Mmcif chain Unbound Bound:G-C-C-A-A-T-T-G-G-C (chain D:double stranded DNA) Unbound Unbound
1d02B Pdbj logo s Rasmollogo id Rasmollogo chain Mmcif id Mmcif chain Unbound Bound:G-C-C-A-A-T-T-G-G-C (chain C:double stranded DNA) Unbound Unbound

Reference for Active-site residues
resource references E.C.
Swiss-prot;P00624

Active-site residues
PDB Catalytic residues Cofactor-binding residues Modified residues Main-chain involved in catalysis Comment
1eriA Pdbj logo s Rasmollogo id Rasmollogo chain Mmcif id Mmcif chain LYS 113 ASP 91;GLU 111(two Mg2+ binding)
1qc9A Pdbj logo s Rasmollogo id Rasmollogo chain Mmcif id Mmcif chain LYS 113 ASP 91;GLU 111(two Mg2+ binding)
1qc9B Pdbj logo s Rasmollogo id Rasmollogo chain Mmcif id Mmcif chain LYS 113 ASP 91;GLU 111(two Mg2+ binding)
1qc9C Pdbj logo s Rasmollogo id Rasmollogo chain Mmcif id Mmcif chain LYS 113 ASP 91;GLU 111(two Mg2+ binding)
1ckqA Pdbj logo s Rasmollogo id Rasmollogo chain Mmcif id Mmcif chain LYS 113 ASP 91;GLU 111(two Mg2+ binding)
1cl8A Pdbj logo s Rasmollogo id Rasmollogo chain Mmcif id Mmcif chain LYS 113 ASP 91;GLU 111(two Mg2+ binding)
1qpsA Pdbj logo s Rasmollogo id Rasmollogo chain Mmcif id Mmcif chain LYS 113 ASP 91;GLU 111(two Mg2+ binding)
1qrhA Pdbj logo s Rasmollogo id Rasmollogo chain Mmcif id Mmcif chain LYS 113 ASP 91;GLU 111(two Mg2+ binding)
1qriA Pdbj logo s Rasmollogo id Rasmollogo chain Mmcif id Mmcif chain LYS 113 ASP 91;GLU 111(two Mg2+ binding)
1d02A Pdbj logo s Rasmollogo id Rasmollogo chain Mmcif id Mmcif chain LYS 100 ;GLU 98(two Mg2+ binding) mutant D83A
1d02B Pdbj logo s Rasmollogo id Rasmollogo chain Mmcif id Mmcif chain LYS 100 ;GLU 98(two Mg2+ binding) mutant D83A

References for Catalytic Mechanism
References Sections No. of steps in catalysis
[6]
Fig.8, Fig.11, p.12-17 2
[7]
Fig.5, p.13492-13494 2
[8]
p.5810-5811
[9]
Fig1, p.6

References
[1]
Resource
Comments X-ray crystallography (3 Angstroms)
Medline ID 87069951
PubMed ID 3024321
Journal Science
Year 1986
Volume 234
Pages 1526-41
Authors McClarin JA, Frederick CA, Wang BC, Greene P, Boyer HW, Grable J, Rosenberg JM
Title Structure of the DNA-Eco RI endonuclease recognition complex at 3 A resolution.
Related PDB
Related UniProtKB P00642
[2]
Resource
Comments mutation analysis
Medline ID 91072344
PubMed ID 2254311
Journal J Biol Chem
Year 1990
Volume 265
Pages 21520-6
Authors Hager PW, Reich NO, Day JP, Coche TG, Boyer HW, Rosenberg JM, Greene PJ
Title Probing the role of glutamic acid 144 in the EcoRI endonuclease using aspartic acid and glutamine replacements.
Related PDB
Related UniProtKB P00642
[3]
Resource
Comments X-ray crystallography (2.7 Angstroms)
Medline ID 90378308
PubMed ID 2399465
Journal Science
Year 1990
Volume 249
Pages 1307-9
Authors Kim YC, Grable JC, Love R, Greene PJ, Rosenberg JM
Title Refinement of Eco RI endonuclease crystal structure: a revised protein chain tracing.
Related PDB
Related UniProtKB P00642
[4]
Resource
Comments molecular dynamics simulations
Medline ID
PubMed ID 7727057
Journal J Biomol Struct Dyn
Year 1994
Volume 12
Pages 487-525
Authors Kumar S, Duan Y, Kollman PA, Rosenberg JM
Title Molecular dynamics simulations suggest that the Eco RI kink is an example of molecular strain.
Related PDB 1eri
Related UniProtKB
[5]
Resource
Comments catalysis
Medline ID
PubMed ID 7607482
Journal Gene
Year 1995
Volume 157
Pages 157-62
Authors Jeltsch A, Pleckaityte M, Selent U, Wolfes H, Siksnys V, Pingoud A
Title Evidence for substrate-assisted catalysis in the DNA cleavage of several restriction endonucleases.
Related PDB
Related UniProtKB
[6]
Resource
Comments catalysis
Medline ID
PubMed ID 9210460
Journal Eur J Biochem
Year 1997
Volume 246
Pages 1-22
Authors Pingoud A, Jeltsch A
Title Recognition and cleavage of DNA by type-II restriction endonucleases.
Related PDB
Related UniProtKB
[7]
Resource
Comments X-ray crystallography (2.15 Angstroms)
Medline ID
PubMed ID 9811827
Journal Proc Natl Acad Sci U S A
Year 1998
Volume 95
Pages 13489-94
Authors Horton NC, Newberry KJ, Perona JJ
Title Metal ion-mediated substrate-assisted catalysis in type II restriction endonucleases.
Related PDB 1bss
Related UniProtKB
[8]
Resource
Comments X-ray crystallography (1.7 Angstroms)
Medline ID
PubMed ID 10545092
Journal EMBO J
Year 1999
Volume 18
Pages 5805-16
Authors Deibert M, Grazulis S, Janulaitis A, Siksnys V, Huber R
Title Crystal structure of MunI restriction endonuclease in complex with cognate DNA at 1.7 A resolution.
Related PDB 1d02
Related UniProtKB
[9]
Resource
Comments
Medline ID
PubMed ID 10739241
Journal Protein Sci
Year 2000
Volume 9
Pages 1-9
Authors Dall'Acqua W, Carter P
Title Substrate-assisted catalysis: molecular basis and biological significance.
Related PDB
Related UniProtKB

Comments
These enzymes, EcoRI(PDB; 1eri, 1qc9) and MunI(PDB; 1d02) belong to the type II restriction endonucleases.
According to the paper [6], cleavage of DNA by restriction endonucleases yields 3'-OH and 5'-phosphate ends, where hydrolysis of the phosphodiester bonds by EcoRI and EcoRV occurs with inversion of configuration at the phosphorous atom, suggesting an attack of a water molecule in line with the 3'-OH leaving group. In general, hydrolysis of phosphodiester bonds requires three functional entities as follows [6]:
(1) A general base that activates the attacking nucleophile,
(2) A Lewis acid that stabilizes the extra negative charge in the pentacovalent transition state,
(3) An acid that protonates or stabilizes the leaving group.
The literature [6] also described the two possible catalytic mechanisms, the substrate-assisted catalysis model and the two-metal-ion mechanism, as described in the following paragraph. However, this paper supported the substrate-assisted catalysis model more favorably than the two-metal-ion mechanism.
The substrate-assisted catalysis model: The attacking water molecule is oriented and deprotonated by the next phosphate group 3' to the scissile phosphate. The negative charge of the transition state could be stablized by the Mg2+ ion and the semi-conserved lysine. The metal ion is bound by the two conserved acidc amino acid residues. The 3'-O- leaving group is protonated by a Mg2+-bound water [6].
The two-metal-ion mechanism: A metal ion bound at one site is responsible for charge neutralization at the scissile phosphate. The attacking water is considered to be part of the hydration sphere of a metal ion bound at the second site [6].
The literature [7] suggested another possible mechanism, three-metal ion mechanism for type II restriction endonucleases from the structural data, as follows:
A metal ion at site I ligates through water to the 3'-phosphate. A second inner-sphere water molecule on this metal dissociates to provide the attacking hydroxide ion, and this dissociation is aided by the immediately adjacent Lysine, corresponding to Lys113 in EcoRI. The metal at site III provides stabilization of the incipient negative charge as the transtion state develops. An inner-sphere water on this metal is located within hydrogen-bonding distance of the leaving 3'-oxygen. Thus, the site III metal is suggested to be operative in lowering the pKa of this water, so that it may dissociate to immediately protonate the leaving anion [7]. The site II metal is purely structural [7].
Crystal structures of these type II endonucleases, EcoRV, EcoRI and PvuII bound to DNA show that the relative positions of the scissile and adjacent 3'-phosphates are conserved. Therefore, the two metal ions bound in site I and site III may have similar functions in each of these enzymes [7].
###
In the structures with DNA substrate (PDB; 1eri), acidic residues at the active site seems to be disoriented. However, the pattern of the active site structure of 1eri (PDB) is similar to those of EcoRV, BglI and PvuII (S00404, S00405, & S00390, respectively in EzCatDB), suggesting a similar catalytic mechanism to those by the enzymes.
###
More recently, several papers including [9] supported the substrate-assisted mechanism for this enzyme and related enzymes (type II restriction enzymes), ruling out the two-metal-ion mechanism. Thus, we concluded that this enzyme adopts the substrate-assisted mechanism with only one metal ion for catalysis (see EcoRV; S00404 in EzCatDB).

Created Updated
2002-09-27 2009-02-26