title: Effects of protonation on the hydrolysis of triphosphate in vacuum and the implications for catalysis by nucleotide hydrolyzing enzymes.
abstract: BACKGROUND
  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. Here, we systematically examine how protonation of the triphosphate affects the mechanism of hydrolysis.
  
  
  RESULTS
  The 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.
  
  
  CONCLUSIONS
  This 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 (-PγO42-). 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.
authors.affiliation: Research Center for Modeling and Simulation (RCMS), National University of Sciences and Technology (NUST), Sector H-12, 44000, Islamabad, Pakistan.
authors.forename: Farooq Ahmad
authors.initials: FA
authors.lastname: Kiani
authors.affiliation: Computational Biochemistry, Interdisciplinary Center for Scientific Computing (IWR), Heidelberg University, Im Neuenheimer Feld 205, D-69120, Heidelberg, Germany. Stefan.Fischer@iwr.uni-heidelberg.de.
authors.forename: Stefan
authors.initials: S
authors.lastname: Fischer
mesh_terms: D017766:Acid Anhydride Hydrolases / Q000737:chemistry / Q000378:metabolism*; D000255:Adenosine Triphosphate / Q000378:metabolism; D055162:Biocatalysis; D020134:Catalytic Domain; D006868:Hydrolysis; D011122:Polyphosphates / Q000378:metabolism*; D011522:Protons; D013816:Thermodynamics; D014618:Vacuum
chemical_list: D011122:Polyphosphates; D011522:Protons; D000255:Adenosine Triphosphate; D017766:Acid Anhydride Hydrolases; C005692:triphosphoric acid
keywords: ATP hydrolysis; ATPase; Computational biochemistry; Concurrent and sequential mechanisms; Enzymatic mechanism; GTPase