Mutual Effects of Cationic Ligands and Substrate on Activity of the Na+-Transporting Pyrophosphatase of Methanosarcina mazei

The PPi-driven sodium pump (membrane pyrophosphatase) of Methanosarcina mazei (Mm-PPase) absolutely requires Na+ and Mg2+ for activity and additionally employs K+ as a modulating cation. Here we explore relationships among Na+, K+, Mg2+, and PPi binding sites by analyzing the dependency of the Mm-PPase PPi-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 PPi hydrolysis indicated that catalysis involves random order binding of two Mg2+ ions and two Na+ ions, and the binding was almost independent of substrate (Mg2PPi 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-Mg2+-substrate complex. The binding of K+ increases, by a factor of 2.5, the catalytic constant, the Michaelis constant, and the Mg2+ 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.