Quaternary Structure and Metal Ion Requirement of Family II Pyrophosphatases from Bacillus subtilis,Streptococcus gordonii, and Streptococcus mutans

• Published in: The Journal of biological chemistry 2001-07, Vol.276 (27), p.24511-24518
• Main Authors: Parfenyev, A N, Salminen, A, Halonen, P, Hachimori, A, Baykov, A A, Lahti, R
• Format: Article
• Language: English
• Subjects: Bacillus subtilis; Bacillus subtilis - enzymology; Calcium Pyrophosphate - metabolism; Cloning, Molecular; Dimerization; Enzyme Activation; Escherichia coli; Kinetics; Magnesium - metabolism; Manganese - metabolism; Protein Conformation; Pyrophosphatase; Pyrophosphatases - chemistry; Pyrophosphatases - genetics; Pyrophosphatases - metabolism; Solubility; Streptococcus - enzymology; Streptococcus gordonii; Streptococcus mutans; Streptococcus mutans - enzymology
• ISSN: 0021-9258; 1083-351X
• DOI: 10.1074/jbc.M101829200

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 n m for Mn 2+ and 10–80 μ m for Mg 2+ ) and two or three moderate affinity sites ( K d ∼ 1 m m 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 m m Mn 2+ , dramatically shifts the monomer ↔ 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.