Ion-Assisted Phosphoryl Transfer Reactions

Mechanistically related enzymes that are ubiquitous in their requirement for a metal ion are those that catalyze phosphoryl group transfer reactions. These enzymes include the (phospho)kinases and phosphatases, which catalyze the transfer of a phosphoryl group (PO3) from the y-position of a nucleotide to an acceptor molecule (the phosphatases are a special case wherein the acceptor is the solvent water) nucleotidyltransferases and nucleases, which catalyze the transfer of a nucleoside phosphodiester to an acceptor molecule (the nucleases and phosphodiesterases are special cases wherein the acceptor is the solvent water) and phosphomutases. In all of these cases, the mechanism of phosphoryl group transfer can occur through one of several postulated pathways. The possible mechanistic extremes are those described as dissociative, analogous to an Sn 1 reaction. 

metaphosphate (PO3) as the leaving group is formed in the transition state. Metaphosphate is formed with a net change in hybridization and planar geometry. This reactive species can then react with the acceptor. In Eq. (2), the nucleophilic acceptor molecule A interacts with the phosphate center, forming a 

A recent kinetic study of the catalysis of hydrolysis of phosphorylated pyri-dines by Mg in aqueous solution as a model for enzymic phosphoryl transfer reactions has been reported by Herschlag and Jencks (23). They have provided evidence that a l(y -10 rate enhancement can be obtained in the presence of Mg +. From the analysis, approximately a l(y -fold rate enhancement can be attributed to the greater nucleophilicity of the species Mg(OH)+ compared to 

Steady-state kinetics have been used to determine the kinetic mechanisms of many of these enzymes. The questions that have been primarily addressed are the sequence of steps that occur in substrate binding prior and subsequent to the catalytic reaction and the potential formation of covalent enzyme intermediates. Classical interpretation of kinetic analyses has been the determination of the relevant reactions occurring via a random or an ordered sequential reaction, or if the reaction is a double-displacement or Ping-Pong reaction. In the former case, phosphoryl transfer occurs in the ternary complex that contains enzyme, phosphoryl donor, and phosphoryl acceptor. In the latter case, enzyme reacts with 

A sensitive probe applied to understand the nature of the reaction mechanism of group transfer is the stereochemistry of the overall reaction. The reaction at a phosphoryl center normally is a degenerate question, since a monosubstituted phosphate ester or anhydride is proprochiral at the phosphate center. Phosphate centers at a diester or disubstituted anhydride are prochiral. Two related methods to analyze the stereochemistry at a phosphate center have been developed by the generation of chirality at the phosphorus center. The first approach was developed by Usher et al. (24) and gave rise to the formation of isotopi-cally chiral [ 0, 0]thiophosphate esters and anhydrides (I). Isotopically chiral [ 0, 0, 0]phosphates (II) have also been synthesized and the absolute configurations determined. Two primary problems must first be addressed with respect to both of the methods that have been developed the synthesis of the isotopically pure chiral thiophosphates and phosphates and the analysis of the isotopic chirality of the products. An example of the chiral starting substrates, as developed for ATP, is schematically demonstrated. Ad = adenosine. 

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The intron group I ribozymes feature common secondary structure and reaction pathways. Active sites capable of catalyzing consecutive phosphodi-ester reactions produce properly spliced and circular RNAs. Ribozymes fold into a globular conformation and have solvent-inaccessible cores as quantified by Fe(II)-EDTA-induced free-radical cleavage experiments. The Tetrahy-mem group I intron ribozyme catalyzes phosphoryl transfer between guanosine and a substrate RNA strand—the exon. This ribozyme also has been proposed to use metal ions to assist in proper folding, to activate the nucleophile, and to stabilize the transition state. ... 

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