Effects of protonation on the hydrolysis of triphosphate in vacuum and the implications for catalysis by nucleotide hydrolyzing enzymes.

Farooq Ahmad Kiani,Stefan Fischer

doi:10.1186/s12858-016-0068-7

Abstract

BackgroundNucleoside triphosphate (NTP) hydrolysis is a key reaction in biology. It involves breaking two very stable bonds (one P–O bond and one O–H bond of water), in either a concurrent or a sequential way. Here, we systematically examine how protonation of the triphosphate affects the mechanism of hydrolysis.ResultsThe hydrolysis reaction of methyl triphosphate in vacuum is computed with protons in various numbers and position on the three phosphate groups. Protonation is seen to have a strong catalytic effect, with the reaction mechanism depending highly on the protonation pattern.ConclusionThis dependence is apparently complicated, but is shown to obey a well-defined set of rules: Protonation of the α- and β-phosphate groups favors a sequential hydrolysis mechanism, whereas γ-protonation favors a concurrent mechanism, the two effects competing with each other in cases of simultaneous protonation. The rate-limiting step is always the breakup of the water molecule while it attacks the γ-phosphorus, and its barrier is lowered by γ-protonation. This step has significantly lower barriers in the sequential reactions, because the dissociated γ-metaphosphate intermediate (PγO3 −) is a much better target for water attack than the un-dissociated γ-phosphate (−PγO4 2−). The simple chemical logic behind these rules helps to better understand the catalytic strategy used by NTPase enzymes, as illustrated here for the catalytic pocket of myosin.A set of rules was determined that describes how protonating the phosphate groups affects the hydrolysis mechanism of methyl triphosphate: Protonation of the α- and/or β- phosphate groups promotes a sequential mechanism in which P-O bond breaking precedes the breakup of the attacking water, whereas protonation of the γ-phosphate promotes a concurrent mechanism and lowers the rate-limiting barrier of water breakup. The role played by individual protein residues in the catalytic pocket of triphosphate hydrolysing enzymes can be assigned accordingly.Graphical abstract Electronic supplementary materialThe online version of this article (doi:10.1186/s12858-016-0068-7) contains supplementary material, which is available to authorized users.

Highlights

Nucleoside triphosphate (NTP) hydrolysis is a key reaction in biology
Reaction paths To obtain one concurrent and one sequential reaction path for each of the eight protonation states mentioned above, initial constraints on the atomic coordinates were applied to channel the refinement of the minimum energy paths (MEP) into a corresponding valley of the potential energy surface
12 MEPs could be found on the respective potential energy landscapes: 5 concurrent MEP and 7 sequential MEPs nP = 0 44.0

Summary

Introduction

Nucleoside triphosphate (NTP) hydrolysis is a key reaction in biology It involves breaking two very stable bonds (one P–O bond and one O–H bond of water), in either a concurrent or a sequential way. Nucleoside triphosphate (NTP) hydrolysis [1, 2] is an important enzymatic reaction in biology [3]. 2) A sequential mechanism, in which the Pγ–Oβγ bond breaks (Fig. 2c) before the OH− nucleophilic attack (Fig. 2d) [8, 9] In vacuum, both mechanisms have similar high-energy barriers, respectively 44.0 and 45.9 kcal mol−1 for concurrent and sequential reactions. To help understand the catalytic mechanism in NTPase enzymes, we are studying here the triphosphate substrate in vacuum, and investigate in particular how protonation of different phosphate groups affects the hydrolysis reaction

Methods

Results

Discussion

Conclusion