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 18420		15377 15377
PubChem	888 888		22247451 962 22247451 962

1eriA	Unbound	Analogue:T-C-G-C-G-A-A-T-T-C-G-C-G (chain B)		Unbound	Unbound
1qc9A	Unbound	Unbound		Unbound	Unbound
1qc9B	Unbound	Unbound		Unbound	Unbound
1qc9C	Unbound	Unbound		Unbound	Unbound
1ckqA	Unbound	Bound:T-C-G-C-G-A-A-T-T-C-G-C-G (chain B)		Unbound	Unbound
1cl8A	Unbound	Bound:T-C-G-C-G-A-PRN-T-T-C-G-C-G (chain B)		Unbound	Unbound
1qpsA	Analogue:_MN	Unbound		Bound:A-A-T-T-C-G-C-G (chain N)	Bound:T-C-G-C-G (chain M)
1qrhA	Unbound	Bound:T-C-G-C-G-A-A-T-T-C-G-C-G (chain M)		Unbound	Unbound
1qriA	Unbound	Bound:T-C-G-C-G-A-A-T-T-C-G-C-G (chain M)		Unbound	Unbound
1d02A	Unbound	Bound:G-C-C-A-A-T-T-G-G-C (chain D:double stranded DNA)		Unbound	Unbound
1d02B	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	LYS 113	ASP 91;GLU 111(two Mg2+ binding)
1qc9A	LYS 113	ASP 91;GLU 111(two Mg2+ binding)
1qc9B	LYS 113	ASP 91;GLU 111(two Mg2+ binding)
1qc9C	LYS 113	ASP 91;GLU 111(two Mg2+ binding)
1ckqA	LYS 113	ASP 91;GLU 111(two Mg2+ binding)
1cl8A	LYS 113	ASP 91;GLU 111(two Mg2+ binding)
1qpsA	LYS 113	ASP 91;GLU 111(two Mg2+ binding)
1qrhA	LYS 113	ASP 91;GLU 111(two Mg2+ binding)
1qriA	LYS 113	ASP 91;GLU 111(two Mg2+ binding)
1d02A	LYS 100	;GLU 98(two Mg2+ binding)			mutant D83A
1d02B	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).

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