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 be 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 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 degrees 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 Mn2+. The dimers were found to have one high affinity manganese-specific site (K(d) of 0.2-3 nm for Mn2+ and 10-80 microm for Mg2+) and two or three moderate affinity sites (K(d) approximately 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 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.
Parfenyev
A N
AN
A. N. Belozersky Institute of Physico-Chemical Biology and School of Chemistry, Moscow State University, Moscow 119899, Russia.
Salminen
A
A
Halonen
P
P
Hachimori
A
A
Baykov
A A
AA
Lahti
R
R
D001412:Bacillus subtilis / Q000201:enzymology*; D002131:Calcium Pyrophosphate / Q000378:metabolism; D003001:Cloning, Molecular; D019281:Dimerization; D004789:Enzyme Activation; D004926:Escherichia coli; D007700:Kinetics; D008274:Magnesium / Q000378:metabolism; D008345:Manganese / Q000378:metabolism; D011487:Protein Conformation; D011755:Pyrophosphatases / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D012995:Solubility; D013291:Streptococcus / Q000201:enzymology*; D013295:Streptococcus mutans / Q000201:enzymology
D008345:Manganese; D011755:Pyrophosphatases; D008274:Magnesium; D002131:Calcium Pyrophosphate