Nucleotidyl transfer transition states

Yet another super family, the nucleotidyl-transferase family, also utilizes two-metal-ion-dependent catalysis the members include transposases, retrovirus integrases and Holliday junction resolvases4. Whereas in the nucleases, the Mg2+ ions are asymmetrically coordinated, and play distinct roles, in activating the nucleophile and stabilizing the transition state, respectively, in the transposases, they are symmetrically coordinated and exchange roles to alternatively activate a water molecule and a 3 -OH for successive strand cleavage and transfer. 

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). 

Very little is known about the transition states for enzymic nucleotidyl and phospho transfer reactions. Inasmuch as the mechanisms of the nonenzymic sol-volyses of phosphate esters establish the possibilities for comparable enzymic reactions, either associative or dissociative mechanisms can be considered. However, the dissociative mechanism is thought to be possible only for phospho transfer reactions and not for nucleotidyl transfers, because metaphosphate cannot be formed from diesters. A dissociative mechanism for a diester would entail the formation of a monomeric metaphosphate monoester, a species that can exist but which has not been observed in solvolysis reactions. The difficulty with the dissociative mechanism for phosphodiesters may be that a monoanion cannot provide enough driving force to expel a leaving group under solvolytic conditions. 

Castro C, Smidansky E, Maksimchuk KR, Arnold JJ, Korneeva VS, Gone M, Konigsberg W, Cameron CE (2007) Two proton transfers in the transition state for nucleotidyl transfer catalyzed by RNA- and DNA-dependent RNA and DNA polymerases. Proc Natl Acad Sci USA 104 4267... 

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