Heikinheimo P, Lehtonen J, Baykov A, Lahti R, Cooperman B S, Goldman A
Turku Centre for Biotechnology, Finland.
Structure. 1996 Dec 15;4(12):1491-508. doi: 10.1016/s0969-2126(96)00155-4.
Soluble inorganic pyrophosphatase (PPase), an essential enzyme central to phosphorus metabolism, catalyzes the hydrolysis of the phosphoanhydride bond in inorganic pyrophosphate. Catalysis requires divalent metal ions which affect the apparent pKas of the essential general acid and base on the enzyme, and the pKa of the substrate. Three to five metal ions are required for maximal activity, depending on pH and enzyme source. A detailed understanding of catalysis would aid both in understanding the nature of biological mechanisms of phosphoryl transfer, and in understanding the role of divalent cations. Without a high-resolution complex structure such a model has previously been unobtainable.
We report the first two high-resolution structures of yeast PPase, at 2.2 and 2.0 A resolution with R factors of around 17%. One structure contains the two activating metal ions; the other, the product (MnPi)2 as well. The latter structure shows an extensive network of hydrogen bond and metal ion interactions that account for virtually every lone pair on the product phosphates. It also contains a water molecule/hydroxide ion bridging two metal ions and, uniquely, a phosphate bound to four Mn2+ ions.
Our structure-based model of the PPase mechanism posits that the nucleophile is the hydroxide ion mentioned above. This aspect of the mechanism is formally analogous to the "two-metal ion' mechanism of alkaline phosphatase, exonucleases and polymerases. A third metal ion coordinates another water molecule that is probably the required general acid. Extensive Lewis acid coordination and hydrogen bonds provide charge shielding of the electrophile and lower the pKa of the leaving group. This "three-metal ion' mechanism is in detail different from that of other phosphoryl transfer enzymes, presumably reflecting how ancient the reaction is.
可溶性无机焦磷酸酶（PPase）是磷代谢的关键酶，催化无机焦磷酸中磷酸酐键的水解。催化反应需要二价金属离子，这些离子会影响酶上必需的广义酸碱的表观pKa以及底物的pKa。根据pH值和酶来源的不同，最大活性需要三到五个金属离子。深入了解催化作用将有助于理解磷酰基转移的生物学机制的本质，以及二价阳离子的作用。此前，由于缺乏高分辨率的复合物结构，这样的模型无法获得。
我们报告了酵母PPase的前两个高分辨率结构，分辨率分别为2.2 Å和2.0 Å，R因子约为17%。其中一个结构包含两个激活金属离子；另一个结构还包含产物（MnPi）2。后一个结构显示出广泛的氢键和金属离子相互作用网络，几乎覆盖了产物磷酸盐上的每一个孤对电子。它还包含一个连接两个金属离子的水分子/氢氧根离子，以及独特的与四个Mn2+离子结合的磷酸盐。
我们基于结构的PPase机制模型假定亲核试剂是上述氢氧根离子。该机制的这一方面在形式上类似于碱性磷酸酶、核酸外切酶和聚合酶的“双金属离子”机制。第三个金属离子配位另一个可能是所需广义酸的水分子。广泛的路易斯酸配位和氢键为亲电试剂提供电荷屏蔽，并降低离去基团的pKa。这种“三金属离子”机制与其他磷酰基转移酶的机制在细节上不同，大概反映了该反应的古老程度。
