Soluble inorganic pyrophosphatases (PPases) form two nonhomologous families, denoted I and II, that have similar active-site structures but different catalytic activities and metal cofactor specificities. Family II PPases, which are often found in pathogenic bacteria, are more active than family I PPases, and their best cofactor is Mn2+ rather than Mg2+, the preferred cofactor of family I PPases. Here, we present results of a detailed kinetic analysis of a family II PPase from Streptococcus gordonii (sgPPase), which was undertaken to elucidate the factors underlying the different properties of family I and 11 PPases. We measured rates of PPi hydrolysis, PPi synthesis, and P-i/water oxygen exchange catalyzed by sgPPase with Mn2+, Mg2+, or Co2+ in the high-affinity metal-binding site and Mg2+ in the other sites, as well as the binding affinities for several active-site ligands (metal cofactors, fluoride, and P-i). On the basis of these data, we deduced a minimal four-step kinetic scheme and evaluated microscopic rate constants for all eight relevant reaction steps. Comparison of these results with those obtained previously for the well-known family I PPase from Saccharomyces cerevisiae (Y-PPase) led to the following conclusions: (a) catalysis by sgPPase does not involve the enzyme-PPi complex isomerization known to occur in family I PPases, (b) the values of k(cat) for the magnesium forms of sgPPase and Y-PPase are similar because of similar rates of bound PPi hydrolysis and product release, (c) the marked acceleration of sgPPase catalysis in the presence of Mn2+ and Co2+ results from a combined effect of these ions on bound PPi hydrolysis and P-i release, (d) sgPPase exhibits lower affinity for both PPi and P-i, and (e) sgPPase and Y-PPase exhibit similar values of k(cat)/K-m, which characterizes the PPase efficiency in vivo (i.e., at nonsaturating PPi concentrations).
2004. Vol. 43, no 4, 1065-1074 p.