Chapter Four - A Simple Strategy to Determine the Dependence of Membrane-Bound Pyrophosphatases on K⁺ as a Cofactor

Membrane-bound pyrophosphatases (mPPases) couple pyrophosphate hydrolysis to H+and/or Na+pumping across membranes and are found in all domains of life except for multicellular animals including humans. They are important for development and stress resistance in plants. Furthermore, mPPases play a role in virulence of human pathogens that cause severe diseases such as malaria and African sleeping sickness. Sequence analysis, functional studies, and recently solved crystal structures have contributed to the understanding of the mPPase catalytic cycle. However, several key mechanistic features remain unknown. During evolution, several subgroups of mPPases differing in their pumping specificity and cofactor dependency arose. mPPases are classified into one of five subgroups, usually by sequence analysis. However, classification based solely on sequence has been inaccurate in several instances due to our limited understanding of the molecular mechanism of mPPases. Thus, pumping specificity and cofactor dependency of mPPases require experimental confirmation. Here, we describe a simple method for the determination of K+dependency in mPPases using a hydrolytic activity assay. By coupling these dependency studies with site-directed mutagenesis, we have begun to build a better understanding of the molecular mechanisms of mPPases. We optimized the assay for thermostable mPPases that are commonly used as model systems in our lab, but the method is equally applicable to mesophilic mPPases with minor modifications.