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<p>Inorganic pyrophosphatases are essential metalloenzymes for phosphate metabolism. Bacterial Family II Inorganic pyrophosphatases utilize a trinuclear metal center and exhibit higher catalytic activity than binuclear counterparts. Here we show the mechanism underlying this enhanced hydrolytic efficiency in the enzyme from <i>Shewanella</i> sp. AS-11 using X-ray absorption spectroscopy, site-directed mutagenesis, and density functional theory calculations. We identify a catalytic μ<sub>3</sub>-oxo nucleophile—generated by proton transfer from a bridging μ<sub>3</sub>-hydroxide to Asp14—as the key reactive species for hydrolysis. Rotation of Asp14 drives this conversion and constitutes the rate-limiting step, with an activation barrier of 15.5 kcal mol<sup>-1</sup>. The trinuclear metal center promotes hydrolysis by lowering the p<i>K</i><sub>a</sub> of the hydroxide to facilitate μ<sub>3</sub>-oxo formation, stabilizing this intermediate, positioning the nucleophile for optimal in-line attack, and enhancing phosphorus electrophilicity. These findings highlight the importance of reactive species generation and illustrate how metalloenzymes exploit geometric and electronic tuning to achieve high catalytic reactivity.</p><p></p>

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μ₃-Oxo nucleophile formation enables efficient SN2 hydrolysis at the trinuclear metal center in inorganic pyrophosphatase

  • 期刊论文

  • 出版模式：

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  • 发表日期：

    2026年4月2日

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  • Saki Maruoka,
  • Yohei Kametani,
  • Eisuke Magome,
  • Hiroyuki Setoyama,
  • Masahide Kawamoto,
  • Masaki Horitani,
  • Takamasa Teramoto,
  • Yoshimitsu Kakuta,
  • Yoshihito Shiota,
  • Kazunari Yoshizawa,
  • Keiichi Watanabe

摘要

Inorganic pyrophosphatases are essential metalloenzymes for phosphate metabolism. Bacterial Family II Inorganic pyrophosphatases utilize a trinuclear metal center and exhibit higher catalytic activity than binuclear counterparts. Here we show the mechanism underlying this enhanced hydrolytic efficiency in the enzyme from Shewanella sp. AS-11 using X-ray absorption spectroscopy, site-directed mutagenesis, and density functional theory calculations. We identify a catalytic μ3-oxo nucleophile—generated by proton transfer from a bridging μ3-hydroxide to Asp14—as the key reactive species for hydrolysis. Rotation of Asp14 drives this conversion and constitutes the rate-limiting step, with an activation barrier of 15.5 kcal mol-1. The trinuclear metal center promotes hydrolysis by lowering the pKa of the hydroxide to facilitate μ3-oxo formation, stabilizing this intermediate, positioning the nucleophile for optimal in-line attack, and enhancing phosphorus electrophilicity. These findings highlight the importance of reactive species generation and illustrate how metalloenzymes exploit geometric and electronic tuning to achieve high catalytic reactivity.