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The three-dimensional structure of the manganese-phosphate complex of inorganic pyrophosphatase from Succharomyces cerevisiae has been refined to an R factor of 19.0% at 2.4-A resolution. X-ray data were collected from a single crystal using an imaging plate scanner and synchrotron radiation. There is one dimeric omolecule in the asymmetric unit. The upper estimate of the root-mean-square coordinate error is 0.4 A using either the oA plot or the superposition of the two crystallographically independent subunits. The good agreement between the coordinates of the two subunits, which were not subjected to non-crystallographic symmetry restraints, provides independent validation of the structure analysis. The active site in each subunit contains four manganese ions and two phosphates. The manganese ions are coordinated by the side chains of aspartate and glutamate residues. The phosphate groups, which were identified on the basis of their local stereochemistry, interact either directly or via water molecules with manganese ions and lysine, arginine, and tyrosine side chains. The phosphates are bridged by two of the manganese ions. The outer phosphate is exposed to solvent. The inner phosphate is surrounded by all four manganese ions. The ion-binding sites are related to the order of binding previously established from kinetic studies. A hypothesis for the transition state of the catalytic reaction is put forward.

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