Kiani Farooq Ahmad, Fischer Stefan
Research Center for Modeling and Simulation (RCMS), National University of Sciences and Technology (NUST), Sector H-12, 44000, Islamabad, Pakistan.
Computational Biochemistry, Interdisciplinary Center for Scientific Computing (IWR), Heidelberg University, Im Neuenheimer Feld 205, D-69120, Heidelberg, Germany.
BMC Biochem. 2016 Jun 29;17(1):12. doi: 10.1186/s12858-016-0068-7.
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.
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.
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 (PO) is a much better target for water attack than the un-dissociated γ-phosphate (-PO). 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.
核苷三磷酸（NTP）水解是生物学中的关键反应。它涉及以同时或相继的方式断裂两个非常稳定的键（一个P - O键和一个水分子的O - H键）。在此，我们系统地研究三磷酸基团的质子化如何影响水解机制。
在真空中计算了甲基三磷酸在三个磷酸基团上具有不同数量和位置质子时的水解反应。发现质子化具有很强的催化作用，反应机制高度依赖于质子化模式。
这种依赖性显然很复杂，但显示遵循一组明确的规则：α - 和β - 磷酸基团的质子化有利于相继水解机制，而γ - 质子化有利于同时水解机制。在同时质子化的情况下，这两种效应相互竞争。限速步骤始终是水分子攻击γ - 磷时的分解，γ - 质子化降低了其能垒。在相继反应中，这一步骤的能垒显著更低，因为解离的γ - 偏磷酸中间体（PO）比未解离的γ - 磷酸（-PO）更易受到水的攻击。这些规则背后简单的化学逻辑有助于更好地理解NTPase酶所采用的催化策略，如在此以肌球蛋白的催化口袋为例所示。确定了一组规则，描述了磷酸基团的质子化如何影响甲基三磷酸的水解机制：α - 和/或β - 磷酸基团的质子化促进相继机制，其中P - O键断裂先于攻击水分子的分解，而γ - 磷酸的质子化促进同时机制并降低水分子分解的限速能垒。据此可以确定三磷酸水解酶催化口袋中各个蛋白质残基所起的作用。
