Mutual effects of cationic ligands and substrate on activity of the Na+-transporting pyrophosphatase of Methanosarcina mazei.

The PP(i)-driven sodium pump (membrane pyrophosphatase) of Methanosarcina mazei (Mm-PPase) absolutely requires Na(+) and Mg(2+) for activity and additionally employs K(+) as a modulating cation. Here we explore relationships among Na(+), K(+), Mg(2+), and PP(i) binding sites by analyzing the dependency of the Mm-PPase PP(i)-hydrolyzing function on these ligands and protection offered by the ligands against Mm-PPase inactivation by trypsin and the SH-reagent mersalyl. Steady-state kinetic analysis of PP(i) hydrolysis indicated that catalysis involves random order binding of two Mg(2+) ions and two Na(+) ions, and the binding was almost independent of substrate (Mg(2)PP(i) complex) attachment. Each pair of metal ions, however, binds in a positively cooperative (or ordered) manner. The apparent cooperativity is lost only when Na(+) binds to preformed enzyme-Mg(2+)-substrate complex. The binding of K(+) increases, by a factor of 2.5, the catalytic constant, the Michaelis constant, and the Mg(2+) binding affinity, and these effects may result from K(+) binding to either one of the Na(+) sites or to a separate site. The effects of ligands on Mm-PPase inactivation by mersalyl and trypsin are highly correlated and are strongly indicative of ligand-induced enzyme conformational changes. Importantly, Na(+) binding induces a conformational change only when completing formation of the catalytically competent enzyme-substrate complex or a similar complex with a diphosphonate substrate analog. These data indicate considerable flexibility in Mm-PPase structure and provide evidence for its cyclic change in the course of catalytic turnover.