|Protein name||Type-2 restriction enzyme EcoRI||Type-2 restriction enzyme MunI||type II site-specific deoxyribonucleasetype II restriction enzyme|
|Synonyms||R.EcoRIEC 126.96.36.199Type II restriction enzyme EcoRIEndonuclease EcoRI||R.MunIEC 188.8.131.52Type II restriction enzyme MunIEndonuclease MunI|
|Activity||Endonucleolytic cleavage of DNA to give specific double-stranded fragments with terminal 5''-phosphates.||Endonucleolytic cleavage of DNA to give specific double-stranded fragments with terminal 5''-phosphates.|
|Cofactor||Binds 2 magnesium ions per subunit.|
|Compound table: links to PDB-related databases & PoSSuM|
|Type||divalent metal (Ca2+, Mg2+)||nucleic acids||H2O||nucleic acids,phosphate group/phosphate ion||nucleic acids|
| || || || || || || || || || || || || |
|1eriA||Unbound||Analogue:T-C-G-C-G-A-A-T-T-C-G-C-G(chain B)|| ||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|
|References for Catalytic Mechanism|
|References||Sections||No. of steps in catalysis|
|||Fig.8, Fig.11, p.12-17||2|
|Comments||X-ray crystallography (3 Angstroms)|
|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.|
|Journal||J Biol Chem|
|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.|
|Comments||X-ray crystallography (2.7 Angstroms)|
|Authors||Kim YC, Grable JC, Love R, Greene PJ, Rosenberg JM|
|Title||Refinement of Eco RI endonuclease crystal structure: a revised protein chain tracing.|
|Comments||molecular dynamics simulations|
|Journal||J Biomol Struct Dyn|
|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.|
|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.|
|Journal||Eur J Biochem|
|Authors||Pingoud A, Jeltsch A|
|Title||Recognition and cleavage of DNA by type-II restriction endonucleases.|
|Comments||X-ray crystallography (2.15 Angstroms)|
|Journal||Proc Natl Acad Sci U S A|
|Authors||Horton NC, Newberry KJ, Perona JJ|
|Title||Metal ion-mediated substrate-assisted catalysis in type II restriction endonucleases.|
|Comments||X-ray crystallography (1.7 Angstroms)|
|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.|
|Authors||Dall'Acqua W, Carter P|
|Title||Substrate-assisted catalysis: molecular basis and biological significance.|
|These enzymes, EcoRI(PDB; 1eri, 1qc9) and MunI(PDB; 1d02) belong to the type II restriction endonucleases.|
According to the paper , 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 :
(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  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 .
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 .
The literature  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 . The site II metal is purely structural .
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 .
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  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).