Two-metal-ion mechanism

The crystal structure of a CODH/ACS enzyme was reported only in 2002.43,44 It reveals a trio of Fe, Ni, and Cu at the active site (6). The Cu is linked to the Ni atom through two cysteine-S, the Ni being square planar with two terminal amide ligands. Planarity and amide coordination bear some resemblance to the Ni porphinoid in MCR. A two-metal ion mechanism is likely for acetyl CoA synthesis, in which a Ni-bound methyl group attacks an adjacent Cu—CO fragment with formation of a Cu-acyl intermediate. A methylnickel species in CODH/ACS has been identified by resonance Raman spectroscopy.45... 

Steitz , Steitz JA. A general two-metal-ion mechanism for catalytic RNA. Proc Natl Acad Sci USA 1993 90 6498-6502. 

Beese LS, Steitz . Structural basis for the 3 -5 exonuclease activity of Escherichia //DNA polymerase I a two metal ion mechanism. EMBO J 1991 10 25-33. 

Figure 13 7 The two-metal-ion mechanism for polynucleotide polymerases. One metal ion (usually Mg2+) activates the 3 -OH group of the primer terminus and stabilizes one of the partly negatively charged equatorial oxygen atoms of the phosphoryl group, whereas the other binds the phosphoryl oxygen and the oxygen atoms of the pyrophosphate leaving group. The two metal ions are 3.9 A apart. This mechanism fits both RNA and DNA polymerases. [Modified from T. A. Steitz, Nature, Lond. 391,231 (1998).]...

Polynucleotide polymerases, or nucleotidyl transferases, are enzymes that catalyze the template-instructed polymerization of deoxyribo- or ribonu-cleoside triphosphates into polymeric nucleic acid - DNA or RNA. Depending on their substrate specificity, polymerases are classed as RNA- or DNA-dependent polymerases which copy their templates into RNA or DNA (all combinations of substrates are possible). Polymerization, or nucleotidyl transfer, involves formation of a phosphodiester bond that results from nucleophilic attack of the 3 -OH of primer-template on the a-phosphate group of the incoming nucleoside triphosphate. Although substantial diversity of sequence and function is observed for natural polymerases, there is evidence that many employ the same mechanism for DNA or RNA synthesis. On the basis of the crystal structures of polymerase replication complexes, a two-metal-ion mechanism of nucleotide addition was proposed [1] during this two divalent metal ions stabilize the structure and charge of the expected pentacovalent transition state (Figure B.16.1). 

N Strater, WN Lipscomb (1995) Two-metal ion mechanism of bovine lens leucine aminopeptidase active site solvent structure and binding mode of L-leucinal, a gem-diolate transition state analogue, by X-ray crystallography, Biochemistry 34 14792-14800... 

There have been a few reports of first generation coordination complex structural models for the phosphatase enzyme active sites (81,82), whereas there are some examples of ester hydrolysis reactions involving dinuclear metal complexes (83-85). Kim and Wycoff (74) as well as Beese and Steitz (80) have both published somewhat detailed discussions of two-metal ion mechanisms, in connection with enzymes involved in phosphate ester hydrolysis. Compared to fairly simple chemical model systems, the protein active site mechanistic situation is rather more complex, because side-chain residues near the active site are undoubtedly involved in the catalysis, i.e, via acid-base or hydrogenbonding interactions that either facilitate substrate binding, hydroxide nucleophilic attack, or stabilization of transition state(s). Nevertheless, a simple and very likely role of the Lewis-acidic metal ion center is to... 

Sontheimer EJ, Sun S, Piccirilli JA. Metal ion catalysis during splicing of premessenger RNA. Nature 1997 388 801-805. Steitz TA, Steitz JA. A general two-metal-ion mechanism for catalytic RNA. Proc. Natl. Acad. Sci. U.S.A. 1993 90 6498-6502. Yean SL, Wuenschell G, Termini J, Lin RJ. Metal-ion coordination by U6 small nuclear RNA contributes to catalysis in the spliceosome. Nature 2000 408 881-884. 

Stahley MR, Strobel SA. Structural evidence for a two-metal-ion mechanism of group I intron splicing. Science 2005 309 1587-1590. 

Many ribozymes and proteins that catalyze phosphoryl-transfer reactions use a mechanism employing two metal ions, and early on group II introns were hypothesized to use a similar mechanism (17). However, evidence for the existence of two metal ions in the catalytic core was only found very recently (12, 13, 18). A direct Mg + coordination of the pro-Sv oxygen of the first nucleotide of the catalytic triad is evident based on phosphorothioate substitution experiments (18). Recently, an intact group II was successfully crystallized for the first time (12). This structure confirms the metal contact from the first nucleotide of the catalytic triad and shows additional contacts to this metal ion from the second nucleotide of the catalytic triad and from the first nucleotide upstream of the bulge (Fig. 4a). This nucleotide is additionally coordinated to the second Mg + ion in the core. The distance between the two Mg + ions in the crystal stmcture is 3.9 A, which is in agreement with the proposed two-metal-ion mechanism (17). 

The catalytic center of RNAP includes the binding site for the 3 -end of RNA and the insertion site for the incoming rNTP. In the nucleotidyl transfer reaction, the 3 -OH group in the sugar ring of the RNA primer reacts with the a-phosphorous atom of a ribonucleoside triphosphate by nucleophilic attack, then the Pa-Oap bond is broken and pyrophosphate (PPj) is released. Thus, a nucleotidyl addition to the RNA primer is achieved. Structural and biochemical data have shown that the active centers of all polymerases share certain common features a pair of metal ions (normally divalent magnesium ions Mg " and three universally conserved carboxylates. The two-metal-ion mechanism for the nucleotidyl transfer reaction... 

Structural studies as well as sequence comparisons among polymerases strongly suggest the hypothesis that the phosphoryl transfer reaction of all polymerases is catalyzed by a two metal ion mechanism that was originally proposed by analogy to the well studied two metal mechanism in the 3 exonuclease reaction (14). It is perhaps of interest to note that such a mechanism, which involves only the properties of two correctly positioned divalent metal ions, could easily be used by an enzyme made entirely of RNA and thus could function in an all RNA world. The fidelity of DNA synthesis appears to arise from two sources. First, enforced Watson-Crick interactions at the polymerase active site increases the accuracy of the incorporation step (9,13). Second, there is a competitive editing at the 3 exonuclease active site that removes misincorporated nucleotide (3,5). When nucleotides are... 

Despite the variations in the primary structure, the RNase H domains of HIV-1 RTase (92) and MoLV RTase have tertiary structures strikingly similar to that of E. coli RNase H. The structural similarity includes the positions of the conserved acidic active site residues, two metal ion-binding sites, and other residues implicated in hydrophobic and H-bonding interactions (93). RNase H activity is presumed to cleave RNA by the same two-metal ion mechanism that has been postulated for the 3 —> 5 -exonuclease of E. coli DNA Pol I. 

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