The wealth of kinetic and structural information makes inorganic pyrophosphatases (PPases) a good model system to study the details of enzymatic phosphoryl transfer. The enzyme accelerates metal-complexed phosphoryl transfer 10 10 -fold: but how? Our structures of the yeast PPase product complex at 1.15 Å and fluoride-inhibited complex at 1.9 Å visualize the active site in three different states: substrate-bound, immediate product bound, and relaxed product bound. These span the steps around chemical catalysis and provide strong evidence that a water molecule (O nu) directly attacks PPi with a pK a vastly lowered by coordination to two metal ions and D117. They also suggest that a low-barrier hydrogen bond (LBHB) forms between D117 and O nu, in part because of steric crowding by W100 and N116. Direct visualization of the double bonds on the phosphates appears possible. The flexible side chains at the top of the active site absorb the motion involved in the reaction, which may help accelerate catalysis. Relaxation of the product allows a new nucleophile to be generated and creates symmetry in the elementary catalytic steps on the enzyme. We are thus moving closer to understanding phosphoryl transfer in PPases at the quantum mechanical level. Ultra-high resolution structures can thus tease out overlapping complexes and so are as relevant to discussion of enzyme mechanism as structures produced by time-resolved crystallography. Inorganic pyrophosphatases (PPases) catalyze one of the oldest and most common reactions in cells and provide a good system for detailed analysis of enzymatic phosphoryl transfer from polyphosphate to water. The kinetics are well characterized (1, 2) and high-resolution structures are available along the reaction pathway (3). The enzyme accelerates hydrolysis of metal complexed inorganic pyrophosphate by 10 10 compared with the uncatalyzed reaction (1)-but for PPases, as for enzymes in general, the exact source of rate enhancement remains unclear.The original model of catalysis suggested that the mechanism proceeded in four steps with all steps after substrate binding partially rate-determining (1). The nucleophile, which is generated by coordinating a water molecule (O nu ) to two metal ions and which is further strengthened by donating a hydrogen bond to D117, is one key to pyrophosphate hydrolysis in PPases. In addition, the substrate pK a is adjusted by extensive coordination to charged atoms (positively charged side chains and M 2ϩ ; ref.3).Our most recent solution studies (P. Halonen, unpublished data; refs. 2 and 4) indicate that the enzyme-substrate complex (EM 2 :MPPi or EM 2 :M 2 PPi) undergoes isomerization during the catalytic cycle (Scheme 1; ref. 4). In addition, fluoride inhibition studies (4) are consistent with structural studies (3,5) suggesting that the nucleophile is coordinated to D117.We earlier determined the structure of complexes A and E (Scheme 1), but now have direct structural information on the mechanistically key intermediates C and D, as well as much higher res...

We have determined the structures of the wild type and seven active site variants of yeast inorganic pyrophosphatase (PPase) in the presence of Mg2+ and phosphate, providing the first complete structural description of its catalytic cycle. PPases catalyze the hydrolysis of pyrophosphate and require four divalent metal cations for catalysis; magnesium provides the highest activity. The crystal form chosen contains two monomers in the asymmetric unit, corresponding to distinct catalytic intermediates. In the "closed" wild-type active site, one of the two product phosphates has already dissociated, while the D115E variant "open" conformation is of the hitherto unobserved two-phosphate and two-"bridging" water active site. The mutations affect metal binding and the hydrogen bonding network in the active site, allowing us to explain the effects of mutations. For instance, in Y93F, F93 binds in a cryptic hydrophobic pocket in the absence of substrate, preserving hydrogen bonding in the active site and leading to relatively small changes in solution properties. This is not true in the presence of substrate, when F93 is forced back into the active site. Such subtle changes underline how low the energy barriers are between thermodynamically favorable conformations of the enzyme. The structures also allow us to associate metal binding constants to specific sites. Finally, the wild type and the D152E variant contain a phosphate ion adjacent to the active site, showing for the first time how product is released through a channel of flexible cationic side chains.

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