EzCatDB: S00384
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DB codeS00384
RLCP classification1.15.8300.1170 : Hydrolysis
CATH domainDomain : Restriction EndonucleaseCatalytic domain

Enzyme Name

Protein nameType-2 restriction enzyme BamHItype II site-specific deoxyribonuclease
type II restriction enzyme
Type II restriction enzyme BamHI
Endonuclease BamHI
PfamPF02923 (BamHI)
[Graphical view]

UniProtKB:Accession NumberP23940
Entry nameT2BA_BACAM
ActivityEndonucleolytic cleavage of DNA to give specific double-stranded fragments with terminal 5''-phosphates.
Subcellular location
CofactorBinds 2 magnesium ions per subunit.

Compound table: links to PDB-related databases & PoSSuM

CompoundMagnesiumDNAH2ODNA 5'-phosphateDNA
Typedivalent metal (Ca2+, Mg2+)nucleic acidsH2Onucleic acids,phosphate group/phosphate ionnucleic acids




1bamAUnboundUnbound UnboundUnbound
1bhmAUnboundBound:T-A-T-G-G-A-T-C-C-A-T(chain D) UnboundUnbound
1bhmBUnboundBound:T-A-T-G-G-A-T-C-C-A-T-A(chain C) UnboundUnbound
1esgAUnboundAnalogue:T-G-G-A-T-T-C-A(chain C) UnboundUnbound
1esgBUnboundAnalogue:T-G-A-A-T-C-C-A(chain D) UnboundUnbound
2bamAAnalogue:2x_CABound:T-A-T-G-G-A-T-C-C-A-T(chain D) UnboundUnbound
2bamBUnboundBound:T-A-T-G-G-A-T-C-C-A-T-A(chain C) UnboundUnbound
3bamAAnalogue:2x_MNUnbound Bound:G-A-T-C-C-A-T(chain E)Bound:T-A-T-G(chain D)
3bamBUnboundBound:T-A-T-G-G-A-T-C-C-A-T-A(chain C) UnboundUnbound

Active-site residues
literature [2],[6]
pdbCatalytic residuesCofactor-binding residues
1bamAGLU 113
GLU 77;ASP 94;GLU 111(two Mg2+ binding)
1bhmAGLU 113
GLU 77;ASP 94;GLU 111(two Mg2+ binding)
1bhmBGLU 113
GLU 77;ASP 94;GLU 111(two Mg2+ binding)
1esgAGLU 113
GLU 77;ASP 94;GLU 111(two Mg2+ binding)
1esgBGLU 113
GLU 77;ASP 94;GLU 111(two Mg2+ binding)
2bamAGLU 113
GLU 77;ASP 94;GLU 111(two Mg2+ binding)
2bamBGLU 113
GLU 77;ASP 94;GLU 111(two Mg2+ binding)
3bamAGLU 113
GLU 77;ASP 94;GLU 111(two Mg2+ binding)
3bamBGLU 113
GLU 77;ASP 94;GLU 111(two Mg2+ binding)

References for Catalytic Mechanism
ReferencesSectionsNo. of steps in catalysis
[5]Fig.8, Fig.11, p.12-172
[6]Fig.4(b), p.9132

CommentsX-ray crystallography (1.95 Angstroms)
Medline ID94195433
PubMed ID8145855
AuthorsNewman M, Strzelecka T, Dorner LF, Schildkraut I, Aggarwal AK
TitleStructure of restriction endonuclease BamHI and its relationship to EcoRI.
Related UniProtKBP23940
CommentsX-ray crystallography (1.95 Angstroms)
PubMed ID8081758
AuthorsNewman M, Strzelecka T, Dorner LF, Schildkraut I, Aggarwal AK
TitleStructure of restriction endonuclease bamhi phased at 1.95 A resolution by MAD analysis.
Related PDB1bam
CommentsX-ray crystallography (2.2 Angstroms)
PubMed ID7624794
AuthorsNewman M, Strzelecka T, Dorner LF, Schildkraut I, Aggarwal AK
TitleStructure of Bam HI endonuclease bound to DNA: partial folding and unfolding on DNA binding.
Related PDB1bhm
PubMed ID7607482
AuthorsJeltsch A, Pleckaityte M, Selent U, Wolfes H, Siksnys V, Pingoud A
TitleEvidence for substrate-assisted catalysis in the DNA cleavage of several restriction endonucleases.
PubMed ID9210460
JournalEur J Biochem
AuthorsPingoud A, Jeltsch A
TitleRecognition and cleavage of DNA by type-II restriction endonucleases.
CommentsX-ray crystallography (1.8 Angstroms)
Medline ID98455073
PubMed ID9783752
JournalNat Struct Biol
AuthorsViadiu H, Aggarwal AK
TitleThe role of metals in catalysis by the restriction endonuclease BamHI.
Related PDB2bam,3bam
Related UniProtKBP23940
CommentsX-ray crystallography (2.15 Angstroms)
PubMed ID9811827
JournalProc Natl Acad Sci U S A
AuthorsHorton NC, Newberry KJ, Perona JJ
TitleMetal ion-mediated substrate-assisted catalysis in type II restriction endonucleases.
CommentsX-ray crystallography
PubMed ID10882125
JournalMol Cell
AuthorsViadiu H, Aggarwal AK
TitleStructure of BamHI bound to nonspecific DNA: a model for DNA sliding.
Related PDB1esg

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.)


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