X-RAY STRUCTURE OF Mn-PHOSPHATE COMPLEX OF YEAST INORGANIC PYROPHOSPHATASE AT 2.4 A RESOLUTION. B. Vainshtein, E. Harutyunyan, I. Kuranova, Institute of Crystallography, Moscow, Russia V. Lamzyn, Z. Dauter, K. Wilson, EMBL Outstation, Hamburg, Germany

The three-dimensional crystal structure of the manganese-phosphate complex of inorganic pyrophosphatase from Saccharomyces cerevisiae has been refined to an R factor of 19.O % at 2.4 A resoluion. 21721 unique reflections ( I > ( ) with average redundancy 3.4 and 80 % completness were measured from a single crystal using synchrotron radiation and imaging plate. Atomic model of homodimeric molecule including 4496 protein atoms, 8 Mn and 4 phosphate groups and 222 solvent molecules were refined without non-crystallographic symmetry restraints. The estimate of the r.m.s. coordinate error is O.4 A using either the (_A plot or the superposition of the two crystallographically independent subunits. 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 glutamat residues. The phosphate groups, 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. 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.