Zyryanov Anton B, Vener Alexander V, Salminen Anu, Goldman Adrian, Lahti Reijo, Baykov Alexander A
A. N. Belozersky Institute of Physico-Chemical Biology and School of Chemistry, Moscow State University, Moscow 119899, Russia.
Biochemistry. 2004 Feb 3;43(4):1065-74. doi: 10.1021/bi0357513.
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).
可溶性无机焦磷酸酶（PPases）形成两个非同源家族，分别为I和II，它们具有相似的活性位点结构，但催化活性和金属辅因子特异性不同。II型家族PPases常见于致病细菌中，其活性高于I型家族PPases，其最佳辅因子是Mn(2+)，而非I型家族PPases首选的辅因子Mg(2+)。在此，我们展示了对戈登链球菌II型家族PPase（sgPPase）进行详细动力学分析的结果，旨在阐明I型和II型家族PPases不同特性背后的因素。我们测量了sgPPase在高亲和力金属结合位点结合Mn(2+)、Mg(2+)或Co(2+)且其他位点结合Mg(2+)时催化PP(i)水解、PP(i)合成以及P(i)/水氧交换的速率，以及对几种活性位点配体（金属辅因子、氟化物和P(i)）的结合亲和力。基于这些数据，我们推导了一个最小的四步动力学方案，并评估了所有八个相关反应步骤的微观速率常数。将这些结果与先前针对酿酒酵母中著名的I型家族PPase（Y-PPase）所获得的结果进行比较，得出以下结论：（a）sgPPase的催化不涉及I型家族PPases中已知发生的酶-PP(i)复合物异构化；（b）sgPPase和Y-PPase的镁形式的k(cat)值相似，这是因为结合的PP(i)水解和产物释放速率相似；（c）在Mn(2+)和Co(2+)存在下sgPPase催化的显著加速是由于这些离子对结合的PP(i)水解和P(i)释放的综合作用；（d）sgPPase对PP(i)和P(i)的亲和力均较低；（e）sgPPase和Y-PPase表现出相似的k(cat)/K(m)值，该值表征了PPase在体内的效率（即，在非饱和PP(i)浓度下）。
