Altered Mechanisms of Reactions of Phosphate Esters Bridging a Dinuclear Metal Center

Abstract

Kinetic isotope effects in the nucleophile and leaving group were obtained for the reaction of p-nitrophenyl phosphate monoester coordinated to a dinuclear Co(III) complex. The metal complex of the p-nitrophenyl phosphate monoester was found to hydrolyze by a single-step concerted mechanism, with significant nucleophilic participation in the transition state. By contrast, the hydrolysis of uncomplexed p-nitrophenyl phosphate occurs by a very loose transition state with little bond formation to the nucleophile. Previously, the metal complex of the diester methyl-p-nitrophenyl phosphate was found to hydrolyze via a two-step addition-elimination mechanism, in contrast to the concerted hydrolysis mechanism followed by uncomplexed diesters with the p-nitrophenolate leaving group. These results show that coordination to a metal complex can significantly alter the mechanism of phosphoryl transfer.

Experimental and computational analysis of the transition state for ribonuclease A-catalyzed RNA 2′- O -transphosphorylation

“…KIEs are typically measured by competitive methods (12) in which mixtures of substrates containing either 16 O or 18 O at one of the reactive phosphoryl oxygens are analyzed (Fig. 1B).…”

“…The larger rate constant for either the 18 O-or 16 O-containing RNA results in progressive depletion of that isotopologue in the unreacted substrate population. Quantitative analysis of the 18 O/ 16 O ratio as a function of reaction progress allows calculation of the KIE, which is expressed as the ratio of the rate constants for the light and heavy isotopologues ( (12). KIEs reflect both the extent to which the labeled atom participates in the reaction coordinate and differences in vibrations of the labeled atom in the ground state compared with the transition state (14).…”

“…KIEs reflect both the extent to which the labeled atom participates in the reaction coordinate and differences in vibrations of the labeled atom in the ground state compared with the transition state (14). Bond breakage, or a "looser" bonding environment in the transition state relative to the ground state results in faster reactivity for the light isotopologue, and thus an 18 k that is greater than unity (referred to as a normal KIE). Conversely, bond formation or a "stiffer" bonding environment favors the heavier isotopologue, and the observed KIE is less than unity (referred to as an inverse KIE).…”

“…Conversely, bond formation or a "stiffer" bonding environment favors the heavier isotopologue, and the observed KIE is less than unity (referred to as an inverse KIE). The fractionation of 18 O will also depend on proton transfer in both preequilibrium and chemical steps (12), and may offset the contributions of O-P bonding. Additionally, both bonding and bending vibrational modes can influence the observed KIE on the nonbridging oxygens, and these effects may also be offsetting (13).…”

“…Here, we describe coordinated kinetic isotope effect (KIE) analyses, molecular dynamics simulations, and quantum mechanical calculations to model the transition state and mechanism of RNase A. Comparison of the 18 O KIEs on the 2′O nucleophile, 5′O leaving group, and nonbridging phosphoryl oxygens for RNase A to values observed for hydronium-or hydroxide-catalyzed reactions indicate a late anionic transition state. Molecular dynamics simulations using an anionic phosphorane transition state mimic suggest that H-bonding by protonated His12 and Lys41 stabilizes the transition state by neutralizing the negative charge on the nonbridging phosphoryl oxygens.…”