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

Authors:
Open Access
Publisher website
Google Scholar
Abstract
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 μ 3 -oxo nucleophile—generated by proton transfer from a bridging μ 3 -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 p K a of the hydroxide to facilitate μ 3 -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.
Funding Information
  • Japan Society for the Promotion of Science (JP23KJ1741)
  • Japan Society for the Promotion of Science (JP23K26836)
  • Japan Society for the Promotion of Science (JP24K21245)
  • Japan Society for the Promotion of Science (JP25K08678)

This publication has 44 references indexed in Scilit: