Quaternary structure and metal ion requirement of family II pyrophosphatases from Bacillus subtilis, Streptococcus gordonii, and Streptococcus mutans

Pyrophosphatase (PPase) from Bacillus subtilis has recently been found to b e the first example of a family II soluble PPase with a unique requirement for Mn2+. In the present work, we cloned and overexpressed in Escherichia c oli 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 degreesC in comparison with 200-400 s(-1) for family I). A ll three family II PPases were found to be dimeric manganese metalloenzymes , dissociating into much less active monomers upon removal of Mn2+. The dim ers were found to have one high affinity manganese-specific site (K-d of 0. 2-3 nM for Mn2+ and 10-80 muM for Mg2+) and two or three moderate affinity sites (K-d, similar to 1 mM for both cations) per subunit, Mn2+ binding to the high affinity site, which occurs with a half-time of less than 10 s at 1.5 mM Mn2+, dramatically shifts the monomer <----> dimer equilibrium in th e direction of the dimer, further activates the dimer, and allows substanti al activity (60-180 s(-1)) against calcium pyrophosphate, a potent inhibito r of family I PPases.