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Our structure-based model of the PPase mechanism posits that the nucleophile is the hydroxide ion mentioned above. This aspect of the mechanism is formally analogous to the "two-metal ion' mechanism of alkaline phosphatase, exonucleases and polymerases. A third metal ion coordinates another water molecule that is probably the required general acid. Extensive Lewis acid coordination and hydrogen bonds provide charge shielding of the electrophile and lower the pKa of the leaving group. This "three-metal ion' mechanism is in detail different from that of other phosphoryl transfer enzymes, presumably reflecting how ancient the reaction is.

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Regulatory CBS (cystathionine β-synthase) domains exist as two or four tandem copies in thousands of cytosolic and membrane-associated proteins from all kingdoms of life. Mutations in the CBS domains of human enzymes and membrane channels are associated with an array of hereditary diseases. Four CBS domains encoded within a single polypeptide or two identical polypeptides (each having a pair of CBS domains at the subunit interface) form a highly conserved disk-like structure. CBS domains act as autoinhibitory regulatory units in some proteins and activate or further inhibit protein function upon binding to adenosine nucleotides (AMP, ADP, ATP, S-adenosyl methionine, NAD, diadenosine polyphosphates). As a result of the differential effects of the nucleotides, CBS domain-containing proteins can sense cell energy levels. Significant conformational changes are induced in CBS domains by bound ligands, highlighting the structural basis for their effects.

The DHH superfamily human protein h-prune, a binding partner of the metastasis suppressor nm23-H1, is frequently overexpressed in metastatic cancers. From an evolutionary perspective, h-prune is very close to eukaryotic exopolyphosphatases. Here, we show for the first time that h-prune efficiently hydrolyzes short-chain polyphosphates (k cat of 3-40 s (-1)), including inorganic tripoly- and tetrapolyphosphates and nucleoside 5'-tetraphosphates. Long-chain inorganic polyphosphates (>or=25 phosphate residues) are converted more slowly, whereas pyrophosphate and nucleoside triphosphates are not hydrolyzed. The reaction requires a divalent metal cofactor, such as Mg (2+), Co (2+), or Mn (2+), which activates both the enzyme and substrate. Notably, the exopolyphosphatase activity of h-prune is suppressed by nm23-H1, long-chain polyphosphates and pyrophosphate, which may be potential physiological regulators. Nucleoside triphosphates, diadenosine hexaphosphate, cAMP, and dipyridamole (inhibitor of phosphodiesterase) do not affect this activity. Mutation of seven single residues corresponding to those found in the active site of yeast exopolyphosphatase led to a severe decrease in h-prune activity, whereas one variant enzyme exhibited enhanced activity. Our results collectively suggest that prune is the missing exopolyphosphatase in animals and support the hypothesis that the metastatic effects of h-prune are modulated by inorganic polyphosphates, which are increasingly recognized as critical regulators in cells.

The active site similiarities, including a water coordinated to two metal ions, suggest that the family II PPase mechanism is "analogous" (not "homologous") to that of family I PPases. This is a remarkable example of convergent evolution. The large change in C-terminal conformation suggests that domain closure might be the mechanism by which Sm-PPase achieves specificity for pyrophosphate over other polyphosphates.

Pyrophosphatase (PPase) from Bacillus subtilis has recently been found to be the first example of a family II soluble PPase with a unique requirement for Mn 2؉ . In the present work, we cloned and overexpressed in Escherichia coli putative genes for two more family II PPases (from Streptococcus mutans and Streptococcus gordonii), isolated the recombinant proteins, and showed them to be highly specific and active PPases (catalytic constants of 1700 -3300 s ؊1 at 25°C in comparison with 200 -400 s ؊1 for family I). All three family II PPases were found to be dimeric manganese metalloenzymes, dissociating into much less active monomers upon removal of Mn 2؉ . The dimers were found to have one high affinity manganese-specific site (K d of 0.2-3 nM for Mn 2؉ and 10 -80 M for Mg 2؉ ) and two or three moderate affinity sites (K d ϳ 1 mM for both cations) per subunit. Mn 2؉ binding to the high affinity site, which occurs with a half-time of less than 10 s at 1.5 mM Mn 2؉ , dramatically shifts the monomer 7 dimer equilibrium in the direction of the dimer, further activates the dimer, and allows substantial activity (60 -180 s ؊1 ) against calcium pyrophosphate, a potent inhibitor of family I PPases.

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...