Dinuclear Zn(II) Complex Catalyzed Phosphodiester Cleavage Proceeds via a Concerted Mechanism: A Density Functional Theory Study

Abstract

Density functional theory (DFT) calculations were used to study the mechanism for the cleavage reaction of the RNA analogue HpPNP (HpPNP = 2-hydroxypropyl-4-nitrophenyl phosphate) catalyzed by the dinuclear Zn(II) complex of 1,3-bis(1,4,7-triazacyclonon-1-yl)-2-hydroxypropane (Zn(2)(L(2)O)). We present a binding mode in which each terminal phosphoryl oxygen atom binds to one zinc center, respectively, and the nucleophilic 2-hydroxypropyl group coordinates to one of the zinc ions, while the hydroxide from deprotonation of a water molecule coordinates to the other zinc ion. Our calculations found a concerted mechanism for the HpPNP cleavage with a 16.5 kcal/mol reaction barrier. An alternative proposed stepwise mechanism through a pentavalent oxyphosphorane dianion reaction intermediate for the HpPNP cleavage was found to be less feasible with a significantly higher energy barrier. In this stepwise mechanism, the deprotonation of the nucleophilic 2-hydroxypropyl group is accompanied with nucleophilic attack in the rate-determining step. Calculations of the nucleophile (18)O kinetic isotope effect (KIE) and leaving (18)O KIE for the concerted mechanism are in reasonably good agreement with the experimental values. Our results indicate a specific-base catalysis mechanism takes place in which the deprotonation of the nucleophilic 2-hydroxypropyl group occurs in a pre-equilibrium step followed by a nucleophilic attack on the phosphorus center. Detailed comparison of the geometric and electronic structure for the HpPNP cleavage reaction mechanisms in the presence/absence of catalyst revealed that the catalyst significantly altered the determining-step transition state to become far more associative or tight, that is, bond formation to the nucleophile was remarkably more advanced than leaving group bond fission in the catalyzed mechanism. Our results are consistent with and provide a reliable interpretation for the experimental observations that suggest the reaction occurs by a concerted mechanism (see Humphry, T.; Iyer, S.; Iranzo, O.; Morrow, J. R.; Richard, J. P.; Paneth, P.; Hengge, A. C. J. Am. Chem. Soc. 2008, 130, 17858-17866) and has a specific-base catalysis character (see Yang, M.-Y.; Iranzo, O.; Richard, J. P.; Morrow, J. R. J. Am. Chem. Soc. 2005, 127, 1064-1065).

Altered (transition) states: mechanisms of solution and enzyme catalyzed RNA 2′-O-transphosphorylation

“…Interestingly, Zn 2 ( 1 ) catalysis of HPpNP transphosphorylation results in a more inverse 18 k NUC (1.0079 vs. 0.9874), while the 18 k LG becomes more normal (1.0064 versus 1.0113) consistent with a later TS[26] (Figure 3B). Recently reported QM calculations obtained assuming coordination of both non-bridging oxygens and the nucleophile by the two Zn 2+ atoms in Zn 2 ( 1 )[40] support a concerted mechanism with a later TS, although one that is more associative relative to the uncatalyzed TS, is consistent with the observed KIEs. In contrast, the distinct dinuclear Zn organometallic complex catalyst, Zn 2 ( 2 ) (Figure 3A) is proposed to act by stabilization of a phosphorane intermediate as evidenced by its ability to catalyze both cleavage and isomerization [37].…”

Isotope effect analyses provide evidence for an altered transition state for RNA 2′-O-transphosphorylation catalyzed by Zn2+

“…The change in the magnitude of these effects reflects an overall later TS with greater nucleophilic bond formation. 45 Recent computational simulations of HPPNP transphosphorylation are consistent with the KIE data and suggest a more associative TS, 46 although one that is still early compared to the TS for RNA transphosphorylation demonstrated here. Nonetheless, a similar theme is observed relevant to enzyme mechanism.…”

QM/MM Analysis Suggests That Alkaline Phosphatase (AP) and Nucleotide Pyrophosphatase/Phosphodiesterase Slightly Tighten the Transition State for Phosphate Diester Hydrolysis Relative to Solution: Implication for Catalytic Promiscuity in the AP Superfamily

“…This can be tested by substituting the zinc ions with other metals of different size (e.g., replacing Zn 2+ by Co 2+ ) and performing kinetic and LFER analyses. Along this line, analysis of TS nature with synthetic analogs of di-metallic zinc motifs 92,93 will be highly informative, since the structure of such artificial catalysts can be better controlled and monitored.…”