DB code: S00383

RLCP classification	1.15.9400.1180 : Hydrolysis
CATH domain	3.40.91.10 : Restriction Endonuclease	Catalytic domain
E.C.	3.1.21.4
CSA	1cfr
M-CSA	1cfr
MACiE

CATH domain	Related DB codes (homologues)

Uniprot Enzyme Name
UniprotKB	Protein name	Synonyms	Pfam
P56200	Type-2 restriction enzyme Cfr10I	R.Cfr10I EC 3.1.21.4 Type II restriction enzyme Cfr10I Endonuclease Cfr10I	PF07832 (Bse634I) [Graphical View]

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

UniprotKB: Accession Number	Entry name	Activity	Subunit	Subcellular location	Cofactor
P56200	T2CX_CITFR	Endonucleolytic cleavage of DNA to give specific double-stranded fragments with terminal 5''-phosphates.			Binds 2 magnesium ions per subunit.

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

1cfrA	Unbound	Unbound		Unbound	Unbound

Reference for Active-site residues
resource	references	E.C.
literature [2]

Active-site residues
PDB	Catalytic residues	Cofactor-binding residues	Modified residues	Main-chain involved in catalysis	Comment

1cfrA	LYS 190	GLU 71;ASP 134;GLU 204(Mg2+ binding)

References for Catalytic Mechanism
References	Sections	No. of steps in catalysis
[2]	p.181-182
[3]	Fig.8, Fig.11, p.12-17	2
[4]	Fig.5, p.13492-13494	2
[5]	Fig.1, p.6

References
[1]
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
[2]
Resource
Comments	X-ray crystallography (2.15 Angstroms)
Medline ID	96144841
PubMed ID	8568865
Journal	J Mol Biol
Year	1996
Volume	255
Pages	176-86
Authors	Bozic D, Grazulis S, Siksnys V, Huber R
Title	Crystal structure of Citrobacter freundii restriction endonuclease Cfr10I at 2.15 A resolution.
Related PDB	1cfr
Related UniProtKB	P56200
[3]
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
[4]
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
Related UniProtKB
[5]
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
This enzyme belongs to the type II restriction endonucleases. Although literature [1] suggests that this enzyme may not employ substrate assistance in catalysis, which is adopted by other type II restriction endonucleases, it is considered to take a similar catalytic mechanism ([2], [3] & [4]). According to the paper [3], 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 [3]: (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 [3] 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 [3]. 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 [3]. The literature [4] 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 residue, coresponding to Lys190 in this enzyme. 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 [4]. The site II metal is purely structural [4]. 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 [4]. ### However, more recent paper [5] supported the substrate-assisted mechanism for the 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). In addition, the pattern of the active site structure is similar to those of EcoRI, EcoRV, BglI and PvuII (S00403, S00404, S00405, & S00390, respectively in EzCatDB), suggesting a similar catalytic mechanism to those by the enzymes, although the structures with ligand molecules are not available.

Comments

This enzyme belongs to the type II restriction endonucleases.
Although literature [1] suggests that this enzyme may not employ substrate assistance in catalysis, which is adopted by other type II restriction endonucleases, it is considered to take a similar catalytic mechanism ([2], [3] & [4]).
According to the paper [3], 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 [3]:
(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 [3] 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 [3].
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 [3].
The literature [4] 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 residue, coresponding to Lys190 in this enzyme. 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 [4]. The site II metal is purely structural [4].
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 [4].
###
However, more recent paper [5] supported the substrate-assisted mechanism for the 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).
In addition, the pattern of the active site structure is similar to those of EcoRI, EcoRV, BglI and PvuII (S00403, S00404, S00405, & S00390, respectively in EzCatDB), suggesting a similar catalytic mechanism to those by the enzymes, although the structures with ligand molecules are not available.

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