THE STRUCTURAL BASIS FOR PYROPHOSPHATASE CATALYSIS

Background: Soluble inorganic pyrophosphatase (PPase), an essential en zyme central to phosphorus metabolism, catalyzes the hydrolysis of the phosphoanhydride bond in inorganic pyrophosphate, Catalysis requires divalent metal ions which affect the apparent pK(a)s of the essential general acid and base on the enzyme, and the pK(a) of the substrate, T hree to five metal ions are required for maximal activity, depending o n pH and enzyme source. A detailed understanding of catalysis would ai d both in understanding the nature of biological mechanisms of phospho ryl transfer, and in understanding the role of divalent cations. Witho ut a high-resolution complex structure such a model has previously bee n unobtainable. Results: We report the first two high-resolution struc tures of yeast PPase, at 2.2 and 2.0 Angstrom resolution with R factor s 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 al so contains a water molecule/hydroxide ion bridging two metal ions and , uniquely, a phosphate bound to four Mn2+ ions. Conclusions: Our stru cture-based model of the PPase mechanism posits that the nucleophile i s the hydroxide ion mentioned above. This aspect of the mechanism is f ormally analogous to the 'two-metal ion' mechanism of alkaline phospha tase, exonucleases and polymerases. A third metal ion coordinates anot her water molecule that is probably the required general acid. Extensi ve Lewis acid coordination and hydrogen bonds provide charge shielding of the electrophile and lower the pK(a) of the leaving group. This 't hree-metal ion' mechanism is in detail different from that of other ph osphoryl transfer enzymes, presumably reflecting how ancient the react ion is.