Admiraal S J, Herschlag D
B400 Beckman Center, Department of Biochemistry, Stanford University, CA 94305-5307, USA.
Chem Biol. 1995 Nov;2(11):729-39. doi: 10.1016/1074-5521(95)90101-9.
Phosphoryl transfer, typically involving high energy phosphate donors such as ATP, is the most common class of biological reactions. Despite this, the transition state for phosphoryl transfer from ATP in solution has not been systematically investigated. Characterization of the transition state for the uncatalyzed hydrolysis of ATP would provide a starting point for dissection of enzyme-catalyzed reactions.
We examined phosphoryl transfer from ATP, GTP and pyrophosphate to a series of alcohols; these reactions are analogous to the phosphorylation of sugars and other biological alcohols and to the hydrolysis of ATP. The Brønsted beta(nucleophile) value of 0.07 is small, indicating that there is little bond formation between the incoming nucleophile and the electrophilic phosphoryl group in the transition state. Coordination of Mg2+ has no measurable effect on this value. The Brønsted beta(leaving group) value of -1.1 for phosphoryl transfer to water from a series of phosphoanhydrides is large and negative, suggesting that the bond between phosphorous and the leaving group oxygen is largely broken in the transition state.
Uncatalyzed hydrolysis of ATP in solution occurs via a dissociative, metaphosphate-like transition state, with little bond formation between nucleophile and ATP and substantial cleavage of the bond between the gamma-phosphoryl moiety and the ADP leaving group. Bound Mg2+ does not perturb the dissociative nature of the transition state, contrary to proposals that enzyme-bound metal ions alter this structure. The simplest expectation for phosphoryl transfer at the active site of enzymes thus entails a dissociative transition state. These results provide a basis for analyzing catalytic mechanisms for phosphoryl transfer.
磷酰基转移是最常见的一类生物反应，通常涉及如ATP等高能量磷酸供体。尽管如此，溶液中ATP磷酰基转移的过渡态尚未得到系统研究。对ATP无催化水解的过渡态进行表征将为剖析酶催化反应提供一个起点。
我们研究了从ATP、GTP和焦磷酸到一系列醇的磷酰基转移；这些反应类似于糖和其他生物醇的磷酸化以及ATP的水解。布朗斯特β（亲核试剂）值为0.07，较小，表明在过渡态中亲核试剂与亲电磷酰基之间几乎没有键的形成。Mg2+的配位对此值没有可测量的影响。从一系列磷酸酐向水进行磷酰基转移的布朗斯特β（离去基团）值为-1.ll，较大且为负，表明在过渡态中磷与离去基团氧之间的键大部分已断裂。
溶液中ATP的无催化水解通过离解的、类似偏磷酸的过渡态发生，亲核试剂与ATP之间几乎没有键的形成，γ-磷酰基部分与ADP离去基团之间的键大量断裂。与酶结合的金属离子会改变这种结构的提议相反，结合的Mg2+不会干扰过渡态的离解性质。因此，对酶活性位点磷酰基转移最简单的预期是离解的过渡态。这些结果为分析磷酰基转移的催化机制提供了基础。
