Rates of elementary catalytic steps for different metal forms of the family II pyrophosphatase from Streptococcus gordonii.
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 Mn(2+) rather than Mg(2+), 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 II PPases. We measured rates of PP(i) hydrolysis, PP(i) synthesis, and P(i)/water oxygen exchange catalyzed by sgPPase with Mn(2+), Mg(2+), or Co(2+) in the high-affinity metal-binding site and Mg(2+) 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-PP(i) 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 PP(i) hydrolysis and product release; (c) the marked acceleration of sgPPase catalysis in the presence of Mn(2+) and Co(2+) results from a combined effect of these ions on bound PP(i) hydrolysis and P(i) release; (d) sgPPase exhibits lower affinity for both PP(i) 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 PP(i) concentrations).
Zyryanov
Anton B
AB
A. N. Belozersky Institute of Physico-Chemical Biology and School of Chemistry, Moscow State University, Moscow 119899, Russia.
Vener
Alexander V
AV
Salminen
Anu
A
Goldman
Adrian
A
Lahti
Reijo
R
Baykov
Alexander A
AA
D001665:Binding Sites; D002118:Calcium / Q000737:chemistry; D002384:Catalysis; D003035:Cobalt / Q000737:chemistry; D011756:Diphosphates / Q000737:chemistry; D043564:Inorganic Pyrophosphatase / Q000737:chemistry* / Q000145:classification; D007700:Kinetics; D008274:Magnesium; D008345:Manganese / Q000737:chemistry; D008670:Metals / Q000737:chemistry*; D010103:Oxygen Isotopes / Q000737:chemistry; D011485:Protein Binding; D029701:Saccharomyces cerevisiae Proteins / Q000737:chemistry / Q000145:classification; D012996:Solutions; D013291:Streptococcus / Q000201:enzymology*; D013379:Substrate Specificity; D014867:Water / Q000737:chemistry
D011756:Diphosphates; D008670:Metals; D010103:Oxygen Isotopes; D029701:Saccharomyces cerevisiae Proteins; D012996:Solutions; D014867:Water; D003035:Cobalt; D008345:Manganese; D043564:Inorganic Pyrophosphatase; D008274:Magnesium; D002118:Calcium