Effect of protonation on the mechanism of phosphate monoester hydrolysis and comparison with the hydrolysis of nucleoside triphosphate in biomolecular motors

Abstract

Hydrolysis of phosphate groups is a crucial reaction in living cells. It involves the breaking of two strong bonds, i.e. the O_aH bond of the attacking water molecule, and the PO_l bond of the substrate (O_a and O_l stand for attacking and leaving oxygen atoms). Mechanism of the hydrolysis reaction can proceed either by a concurrent or a sequential mechanism. In the concurrent mechanism, the breaking of O_aH and PO_l bonds occurs simultaneously, whereas in the sequential mechanism, the O_aH and PO_l bonds break at different stages of the reaction. To understand how protonation affects the mechanism of hydrolysis of phosphate monoester, we have studied the mechanism of hydrolysis of protonated and deprotonated phosphate monoester at M06-2X/6-311+G**//M06-2X/6-31+G*+ZPE level of theory (where ZPE stands for zero point energy). Our calculations show that in both protonated and deprotonated cases, the breaking of the water O_aH bond occurs before the breaking of the PO_l bond. Because the two events are not separated by a stable intermediate, the mechanism can be categorized as semi-concurrent. The overall energy barrier is 41kcalmol^-1 in the unprotonated case. Most (5/6th) of this is due to the initial breaking of the water O_aH bond. This component is lowered from 34 to 25kcalmol^-1 by adding one proton to the phosphate. The rest of the overall energy barrier comes from the subsequent breaking of the PO_l bond and is not sensitive to protonation. This is consistent with previous findings about the effect of triphosphate protonation on the hydrolysis, where the equivalent protonation (on the γ-phosphate) was seen to lower the barrier of breaking the water O_aH bond and to have little effect on the PO_l bond breaking. Hydrolysis pathways of phosphate monoester with initial breaking of the PO_l bond could not be found here. This is because the leaving group in phosphate monoester cannot be protonated, unlike in triphosphate hydrolysis, where protonation of the β- and γ-phosphates had been shown to promote a mechanism where the PO_l bond breaks before the O_aH bond does. We also point out that the charge shift due to PO_l bond breaking during sequential ATP hydrolysis in bio-molecular motors onsets the week unbinding of hydrolysis product that finally leads to the product release during power stroke.

Keywords: Biomolecular motors; Computational biochemistry; Phosphate monoester hydrolysis; Phosphoryl transfer reactions; Reaction mechanism.