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.

Endometrial or endometriotic tissue E2 concentrations are actively regulated by local estrogen metabolism in the tissue. Thus, the inhibition of local E2 synthesis is a valid, novel approach to reduce local E2-dependent growth of endometriotic tissue.

An open reading frame located in the COTF-TETB intergenic region of Bacillus subtilis was cloned and expressed in Escherichia coli and shown to encode inorganic pyrophosphatase (PPase). The isolated enzyme is Mn P+ -activated, like the authentic PPase isolated from B. subtilis. Although 13 functionally important active site residues are conserved in all 31 soluble PPase sequences so far identified, only two of them are conserved in B. subtilis PPase. This suggests that B. subtilis PPase represents a new family of soluble PPases (a Bs family), putative members of which were found in Archaeoglobus fulgidus, Methanococcus jannaschii, Streptococcus mutans and Streptococcus gordonii.z 1998 Federation of European Biochemical Societies.

Based on the primary structure, soluble inorganic pyrophosphatases can be divided into two families which exhibit no sequence similarity to each other. Family I, comprising most of the known pyrophosphatase sequences, can be further divided into prokaryotic, plant and animal/fungal pyrophosphatases. Interestingly, plant pyrophosphatases bear a closer similarity to prokaryotic than to animal/fungal pyrophosphatases. Only 17 residues are conserved in all 37 pyrophosphatases of family I and remarkably, 15 of these residues are located at the active site. Subunit interface residues are conserved in animal/fungal but not in prokaryotic pyrophosphatases.z 1999 Federation of European Biochemical Societies.

Family II inorganic pyrophosphatases (PPases) constitute a new evolutionary group of PPases, with a different fold and mechanism than the common family I enzyme; they are related to the "DHH" family of phosphoesterases. Biochemical studies have shown that Mn(2+) and Co(2+) preferentially activate family II PPases; Mg(2+) partially activates; and Zn(2+) can either activate or inhibit (Zyryanov et al., Biochemistry, 43, 14395-14402, accompanying paper in this issue). The three solved family II PPase structures did not explain the differences between the PPase families nor the metal ion differences described above. We therefore solved three new family II PPase structures: Bacillus subtilis PPase (Bs-PPase) dimer core bound to Mn(2+) at 1.3 A resolution, and, at 2.05 A resolution, metal-free Bs-PPase and Streptococcus gordonii (Sg-PPase) containing sulfate and Zn(2+). Comparison of the new and old structures of various family II PPases demonstrates why the family II enzyme prefers Mn(2+) or Co(2+), as an activator rather than Mg(2+). Both M1 and M2 undergo significant changes upon substrate binding, changing from five-coordinate to octahedral geometry. Mn(2+) and Co(2+), which readily adopt different coordination states and geometries, are thus favored. Combining our structures with biochemical data, we identified M2 as the high-affinity metal site. Zn(2+) activates in the M1 site, where octahedral geometry is not essential for catalysis, but inhibits in the M2 site, because it is unable to assume octahedral geometry but remains trigonal bipyramidal. Finally, we propose that Lys205-Gln81-Gln80 form a hydrophilic channel to speed product release from the active site.

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Forty-two subjects with acute tularemia were studied for the occurrence of C-reactive protein (CRP), and 73 subjects with acute tularemia or experience of the disease within the last 11 years were studied for immunoglobulin M (IgM), IgA, and IgG class-specific antibodies, agglutinating antibodies, and complementfixing antibodies to Francisella tularensis by using an enzyme-linked immunosorbent assay (ELISA), the tube agglutination test, and a complement-fixing ELISA. The incubation time between infection and the outbreak of symptoms varied from 1 to 10 days, averaging 6.5 days. Elevated CRP concentrations were found in all samples taken in the first 6 days of illness, when the antibodies generally were absent. The highest CRP values, up to 165 mg/liter, occurred in the earliest samples and then decreased rapidly, being undetectable (<1 mg/liter) from 1 month after the onset of symptoms. Simultaneous though individually varying formation of IgM, IgA, and IgG class-specific antibodies to F. tularensis was demonstrable by ELISA in all the tularemia patients during the acute stage. In most cases, these antibodies appeared 6 to 10 days after the onset of symptoms, i.e., about 2 weeks after infection, reached their highest values at 4 to 7 weeks, and, despite a decreasing trend in their level, were still present 0.5 to Il years after onset of tularemia, as demonstrable by the agglutination test and by the complement-fixing ELISA. Of the three methods used, ELISA for IgM, IgA, and IgG proved to be the most efficient for the early serodiagnosis of tularemia.