Mechanism of the Nucleotidyl Transfer Reaction

Zhang Y, Zhu R, Zhang R, de la Lande A, Salahub D (2012) On the mechanism of the nucleotidyl transfer reaction catalyzed by yeast RNA polymerase n (Unpublished)... 

Bojin, M.D. and Schlick, T. (2007) A quantum mechanical investigation of possible mechanisms for the nucleotidyl transfer reaction catalyzed by DNA polymerase B. J. Phvs. Chem. B, 111, 11244-11252. 

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... 

This article will consider recent advances in the mechanism of model phosphoryl transfer reactions, magnetic resonance studies of phosphoryl and nucleotidyl transerring enzymes in solution and Xray diffraction studies in the crystalline state which have clarified or at least have suggested the role of the essential divalent cation. 

These enzymes are not classified as nucleotidyltransferases, although they catalyze nucleotidyl group transfers in the course of activating the S -phosphoryl groups for the ligation process. The activation mechanism involves a covalent adenylyl-enzyme as an intermediate and a double displacement on of ATP (or NAD+). The chemical mechanism of the RNA ligase reaction is similar. The stereochemistry of these reactions is known for RNA ligase and is consistent with the mechanism as formulated above (81, 82). 

The rationale for the different mechanisms of the other paired reactions [(36a) and (36b) and (37a) and (37b)] is exactly the same. This pattern is followed by all enzymes of this class. That is, nucleophilic catalysis and covalent intermediates, which are necessary and required for the Ping-Pong kinetic pathway to operate, are found only in those cases involving Ping-Pong kinetics. No phosphotransferase or nucleotidyltransferase is known to catalyze phosphoryl or nucleotidyl group transfer by a sequential kinetic pathway via a doubledisplacement mechanism. However, it is not certain that in all cases of double... 

The role of divalent cations in the mechanism of enzyme catalysed phosphoryl and nucleotidyl transfer reactions. A. S. Mildvan and C. M. Grisham, Struct. Bonding (Berlin), 1974,20,1-21 (88),... 

Studies on the kinetic behaviour of nucleoside and nucleotide complexes are less common than those on structural aspects. This arises because of the rapid rates of the formation and dissociation reactions, requiring NMR or temperature-jump relaxation measurements. The number of species that can coexist in solution also hinders interpretation. The earlier kinetic studies have been reviewed by Frey and Stuehr.127 Two important biological reactions of the nucleotides are phosphoryl and nucleotidyl group transfers. Both reactions are catalytic nucleophilic reactions and they both require the presence of a divalent metal ion, in particular Mg2+. Consequently, one of the main interests has been in understanding the catalytic mechanism of the metal ion involvement. This has mainly involved studies on related non-enzymic reactions.128... 

The mechanisms of nucleotide oligomer synthesis catalyzed by proteinoids have not reported. A tentative mechanism rests on the supposition that the proteinoid nucleotide complex is an intermediate of an enzyme-like nucleotidyl transfer reaction. 

Contents A. S. Mildvan, C. M. Grisham The Role of Divalent Cations in the Mechanism of Enzyme Catalyzed Phosphoryl and Nucleotidyl Transfer Reactions. - H.P.C.Hogenkamp, G.N.Sando The Enzymatic Reduction of Ribonucleotides. - W. T. Oosterhuis The Electronic State of Iron in Some Natural Iron Compounds. Determination by Mossbauer and ESR Spectroscopy. - A. Trautwein Mossbauer Spectroscopy on Heme Proteins. 

The Role of Divalent Cations in the Mechanism of Enzyme Catalyzed Phosphoryl and Nucleotidyl Transfer Reactions ... 

Phosphoryl and nucleotidyl transfer reactions are nucleophilic displacements on phosphorus 3—6), and like analogous displacements on carbon (7) or on metal ions (5), have been found to take place by mechanisms varying between two extreme or limiting cases 1. In the dissociative or SnI mechanism the initial departure of the leaving nucleophile, yields the planar triply coordinate metaphosphate anion as a reactive chemical intermediate (3, 8), which then combines with the entering ligand on either face of the metaphosphate plane 2. In the associative... 

By the criteria of steady-state kinetic patterns and stereochemistry, these enzymes appear to catalyze their respective reactions by similar or closely related mechanisms. The steady-state kinetic mechanisms are of the sequential type, and in all cases so far investigated the reactions proceed with inversion of configuration at P of the nucleoside triphosphate. Thus, these reactions proceed via ternary complexes of enzyme-NTP-ROPOa , and the nucleotidyl transfer is a one-step transfer directly from the NTP to the acceptor, that is, by a single displacement at P of NTP. 

The nucleotidyl transfer step is reaction (29a), which proceeds with inversion of configuration at phosphorus in all of the aminoacyl-tRNA synthetase reactions so far studied [for amino acids (aa) Phe, He, Tyr, and Met] (89-92). Stereochemical inversion shows that the nucleotidyl transfer mechanism involves an uneven number of substitutions on phosphorus. Since no other evidence of an adenylyl-enzyme can be found, aminoacyl activation most likely occurs by a single-displacement mechanism, with direct transfer of the AMP group from ATP to the carboxylate group of the amino acid within the enzyme-amino acid-ATP complex. 

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. 

Fig. 4 The proposed nucleotidyl transfer reaction mechanism. Step 1 The proton of the 3 OH of RNA primer transfers to a solvait water and a proton on water transfers to 02a of a-phosphate simultaneously Step 2 the proton of the 02a atom rotates to the side of P-phosphate Step 3 the 3 0 atom performs a nucleophilic attack at the a-phosphorus atom of the a-phosphate Step 4 the Pa-03p bond of the intermediate cleaves to form a phosphodiester bond and the proton on 02a migrates to 03 P...

We proposed the following detailed nucleotidyl transfer reaction mechanism for yeast RNA polymerase II, shown in Fig. 4. The proton of the 3 OH first transfers to the 02a of a-phosphate via a solvent water molecule, then one of the water molecule s protons transfers to the bridging phosphate 03p atom. Note that the water molecule is located by our CHARMM molecular dynamics (MD) simulations. 

Mildvan and Grisham have reviewed the role of bivalent cations in the mechanism of enzyme-catalysed phosphoryl and nucleotidyl transfer reactions. 

Phosphotransferases and nucleotidyltransferases were last reviewed in this series 15 years ago in Volumes VIII and IX. At that time a major mechanistic question was whether these enzymes catalyze their reactions by single-displacement or double-displacement mechanisms. The two mechanisms differed chemically with respect to whether the phosphoryl or nucleotidyl group is transferred directly between two substrates, or whether the group transfer is mediated by a nucleophilic group of the enzyme in a two-step mechanism via a covalent phos-phoenzyme or nucleotidyl-enzyme. 

Two kinds of information about nucleotidyltransferases and phosphotransferases are obtained by use of substrates or substrate analogs with chiral P. The stereochemical course of phosphoryl transfer and nucleotidyl transfer gives important information about the reaction mechanism. If inversion of configuration at phosphorus is observed, it may be concluded that an uneven number of displacements at phosphorus occurs in the reaction mechanism. If retention of configuration at phosphorus is observed, it may be concluded that the mechanism entails an even number of displacements at phosphorus. Inversion corresponds to the single-displacement mechanism of Eq. (2), and retention indicates a mechanism such as that of Eq. (3) or Eqs. (4a) and (4b). 

The DNA and RNA polymerase reactions, as well as the reverse transcriptase and polynucleotide phosphorylase reactions, proceed with inversion of configuration at Pa of the nucleoside triphosphate (45-50). Thus, an uneven number of displacements at phosphoms is involved in the chemical reaction mechanism, and the stereochemistry provides no evidence for the involvement of a covalent nucleotidyl-enzyme as an intermediate on the catalytic pathway. No other evidence for such an intermediate is available. Therefore, it must be concluded that the physicochemical requirements for nucleotidyl group transfer, substrate recognition, and movement along the template are derived fiom binding interactions between the enzyme and its template and substrate rather than through nucleophilic catalysis. This is also true of polynucleotide phosphorylase and other nucleotidyltransferases that catalyze reactions of polynucleotides (51, 52). 

In contrast to nucleic acid polymerases, polynucleotide processing enzymes often act by mechanisms that involve covalent polynucleotide enzymes as compulsory intermediates (53, 54). The covalent linkages are through phosphodies-ters comprising an enzymic nucleophile, usually the phenolic group of tyrosine, and a nucleotidyl moiety of the nucleic acid. These enzymes are not classified as nucleotidyltransferases, but they catalyze nucleotidyl group transfer as the basic reaction in isomerization processes. Examples are topoisomerases and site-specific recombinases. These enzymes utilize the enzymic nucleophile to cleave the polynucleotide in such a way as to preserve the energy of the covalent bond... 

The most general mechanism for the synthesis of sugar nucleotides, first elucidated by Munch-Petersen and coworkers, involves the transfer of a nucleotidyl group from a nucleoside triphosphate to a glycosyl phosphate with the concomitant release of pyrophosphate. For instance, uridine 5-(a-D-glucopyranosyl pyrophosphate) is formed in numerous organ-isms by the following reaction. 

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