DB code: S00384

RLCP classification	1.15.8300.1170 : Hydrolysis
CATH domain	3.40.91.20 : Restriction Endonuclease	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
P23940	Type-2 restriction enzyme BamHI	R.BamHI EC 3.1.21.4 Type II restriction enzyme BamHI Endonuclease BamHI	PF02923 (BamHI) [Graphical View]

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

UniprotKB: Accession Number	Entry name	Activity	Subunit	Subcellular location	Cofactor
P23940	T2BA_BACAM	Endonucleolytic cleavage of DNA to give specific double-stranded fragments with terminal 5''-phosphates.	Homodimer.		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

1bamA	Unbound	Unbound		Unbound	Unbound
1bhmA	Unbound	Bound:T-A-T-G-G-A-T-C-C-A-T (chain D)		Unbound	Unbound
1bhmB	Unbound	Bound:T-A-T-G-G-A-T-C-C-A-T-A (chain C)		Unbound	Unbound
1esgA	Unbound	Analogue:T-G-G-A-T-T-C-A (chain C)		Unbound	Unbound
1esgB	Unbound	Analogue:T-G-A-A-T-C-C-A (chain D)		Unbound	Unbound
2bamA	Analogue:2x_CA	Bound:T-A-T-G-G-A-T-C-C-A-T (chain D)		Unbound	Unbound
2bamB	Unbound	Bound:T-A-T-G-G-A-T-C-C-A-T-A (chain C)		Unbound	Unbound
3bamA	Analogue:2x_MN	Unbound		Bound:G-A-T-C-C-A-T (chain E)	Bound:T-A-T-G (chain D)
3bamB	Unbound	Bound:T-A-T-G-G-A-T-C-C-A-T-A (chain C)		Unbound	Unbound

Reference for Active-site residues
resource	references	E.C.
literature [2],[6]

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

1bamA	GLU 113	GLU 77;ASP 94;GLU 111(two Mg2+ binding)
1bhmA	GLU 113	GLU 77;ASP 94;GLU 111(two Mg2+ binding)
1bhmB	GLU 113	GLU 77;ASP 94;GLU 111(two Mg2+ binding)
1esgA	GLU 113	GLU 77;ASP 94;GLU 111(two Mg2+ binding)
1esgB	GLU 113	GLU 77;ASP 94;GLU 111(two Mg2+ binding)
2bamA	GLU 113	GLU 77;ASP 94;GLU 111(two Mg2+ binding)
2bamB	GLU 113	GLU 77;ASP 94;GLU 111(two Mg2+ binding)
3bamA	GLU 113	GLU 77;ASP 94;GLU 111(two Mg2+ binding)
3bamB	GLU 113	GLU 77;ASP 94;GLU 111(two Mg2+ binding)

References for Catalytic Mechanism
References	Sections	No. of steps in catalysis
[3]	p.662
[5]	Fig.8, Fig.11, p.12-17	2
[6]	Fig.4(b), p.913	2

References
[1]
Resource
Comments	X-ray crystallography (1.95 Angstroms)
Medline ID	94195433
PubMed ID	8145855
Journal	Nature
Year	1994
Volume	368
Pages	660-4
Authors	Newman M, Strzelecka T, Dorner LF, Schildkraut I, Aggarwal AK
Title	Structure of restriction endonuclease BamHI and its relationship to EcoRI.
Related PDB
Related UniProtKB	P23940
[2]
Resource
Comments	X-ray crystallography (1.95 Angstroms)
Medline ID
PubMed ID	8081758
Journal	Structure
Year	1994
Volume	2
Pages	439-52
Authors	Newman M, Strzelecka T, Dorner LF, Schildkraut I, Aggarwal AK
Title	Structure of restriction endonuclease bamhi phased at 1.95 A resolution by MAD analysis.
Related PDB	1bam
Related UniProtKB
[3]
Resource
Comments	X-ray crystallography (2.2 Angstroms)
Medline ID
PubMed ID	7624794
Journal	Science
Year	1995
Volume	269
Pages	656-63
Authors	Newman M, Strzelecka T, Dorner LF, Schildkraut I, Aggarwal AK
Title	Structure of Bam HI endonuclease bound to DNA: partial folding and unfolding on DNA binding.
Related PDB	1bhm
Related UniProtKB
[4]
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
[5]
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
[6]
Resource
Comments	X-ray crystallography (1.8 Angstroms)
Medline ID	98455073
PubMed ID	9783752
Journal	Nat Struct Biol
Year	1998
Volume	5
Pages	910-6
Authors	Viadiu H, Aggarwal AK
Title	The role of metals in catalysis by the restriction endonuclease BamHI.
Related PDB	2bam 3bam
Related UniProtKB	P23940
[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
Related UniProtKB
[8]
Resource
Comments	X-ray crystallography
Medline ID
PubMed ID	10882125
Journal	Mol Cell
Year	2000
Volume	5
Pages	889-95
Authors	Viadiu H, Aggarwal AK
Title	Structure of BamHI bound to nonspecific DNA: a model for DNA sliding.
Related PDB	1esg
Related UniProtKB

Comments
This enzyme belongs to the type II restriction endonucleases. According to the paper [5], 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 [5]: (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 [5] 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 [5]. 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 [5]. The literature [6] proposed a two-metal mechanism of DNA cleavage for BamHI which is distinct from that of other endonucleases such as EcoRV, instead, similar to that proposed for the 3'-5'-exonuclease domain of the E. coli DNA polymerase I (see M00055-[8]). BamHI is the only restriction endonuclease to be characterized structurally that has a glutamic acid (Glu113) in the position corresponding to a lysine in the others [6]. Glu113 appears to act as a base in accepting a proton from the attacking water [6]. A metal-bound water molecule has a lower pKa value (11.4 for Mg2+, 10.6 for Mn2+) than bulk water, favoring the formation of a nucleophilic hydroxyl species that can attack the scissile phosphodiester. The metal ions are in positions to stabilize the 'entering' and 'leaving' oxygens at the apical positions, and to reduce the energy in forming the 90 degree O-P-O bond angles between the apical and the equatorial oxygens [6]. The mechanism of Ca2+ inhibition is also described in the literature [6]. Firstly, pKa of a Ca2+ bound water (pKa=12.9) is higher than that for Mg2+ (pKa=11.4) or Mn2+ (pKa=10.6), which will have the effect of lowering the concentration of metal-hydroxyl ions for the nucleophilic attack on the scissile phosphodiester. Secondly, Ca2+ is a bulkier ion (radius 0.99 A) than Mg2+ (0.65 A) or Mn2+ (0.80), even compared to other ions (Co2+, Zn2+, & Cd2+). Although the two Ca2+ ions appear to be well accommodated in the ground state of the BamHI-DNA complex, this may not be the case during the transition state. The transition state may cause steric interference with the larger Ca2+ ions. Considering structure with dna and analogous ions (Ca2+), the reaction proceeds as follows: (1) Glu113 acts as a general base to activate a water, which is bound to magnesium ion. (This ion is bound to Glu77 and Asp94.) (2) The activated water makes a nucleophilic attack on the phoshoryc ester of DNA. (3) A pentacovalent transition-state is stabilized by the two magnesium ions. These ions interact with a non-bridging phosphate oxygen and the leaving 3'-oxygen. (4) 3'-end is protonated by another water, which is bound to the other magnesium ion. (This ion is bound to Asp94 and Glu77.)

Comments

This enzyme belongs to the type II restriction endonucleases.
According to the paper [5], 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 [5]:
(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 [5] 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 [5].
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 [5].
The literature [6] proposed a two-metal mechanism of DNA cleavage for BamHI which is distinct from that of other endonucleases such as EcoRV, instead, similar to that proposed for the 3'-5'-exonuclease domain of the E. coli DNA polymerase I (see M00055-[8]).
BamHI is the only restriction endonuclease to be characterized structurally that has a glutamic acid (Glu113) in the position corresponding to a lysine in the others [6]. Glu113 appears to act as a base in accepting a proton from the attacking water [6].
A metal-bound water molecule has a lower pKa value (11.4 for Mg2+, 10.6 for Mn2+) than bulk water, favoring the formation of a nucleophilic hydroxyl species that can attack the scissile phosphodiester. The metal ions are in positions to stabilize the 'entering' and 'leaving' oxygens at the apical positions, and to reduce the energy in forming the 90 degree O-P-O bond angles between the apical and the equatorial oxygens [6].
The mechanism of Ca2+ inhibition is also described in the literature [6]. Firstly, pKa of a Ca2+ bound water (pKa=12.9) is higher than that for Mg2+ (pKa=11.4) or Mn2+ (pKa=10.6), which will have the effect of lowering the concentration of metal-hydroxyl ions for the nucleophilic attack on the scissile phosphodiester. Secondly, Ca2+ is a bulkier ion (radius 0.99 A) than Mg2+ (0.65 A) or Mn2+ (0.80), even compared to other ions (Co2+, Zn2+, & Cd2+). Although the two Ca2+ ions appear to be well accommodated in the ground state of the BamHI-DNA complex, this may not be the case during the transition state. The transition state may cause steric interference with the larger Ca2+ ions.
Considering structure with dna and analogous ions (Ca2+), the reaction proceeds as follows:
(1) Glu113 acts as a general base to activate a water, which is bound to magnesium ion. (This ion is bound to Glu77 and Asp94.)
(2) The activated water makes a nucleophilic attack on the phoshoryc ester of DNA.
(3) A pentacovalent transition-state is stabilized by the two magnesium ions. These ions interact with a non-bridging phosphate oxygen and the leaving 3'-oxygen.
(4) 3'-end is protonated by another water, which is bound to the other magnesium ion. (This ion is bound to Asp94 and Glu77.)

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