Identification of Essential Lysines Involved in Substrate Binding of Vacuolar H+-Pyrophosphatase

Substrate-induced Changes in Domain Interaction of Vacuolar H+-Pyrophosphatase

“…Enzyme Assay and Protein Determination-PP i hydrolysis activity was determined by measuring the release of P i from PP i as delineated previously (7,12,13,26). The reaction medium contained 30 mM Tris/Mes (pH 8.0), 1 mM MgSO 4 , 0.5 mM NaF, 50 mM KCl, 1 mM PP i , 1.5 g/ml gramicidin D, and 20 -30 g/ml microsomal protein.…”

“…Mung bean (V. radiata L.) H ϩ -PPase cDNA (VPP; accession number P21616) was cloned into the Escherichia coli/Saccharomyces cerevisiae shuttle vector, pYES2 (Invitrogen), and the C terminus was fused with His 6 tag (pYVH6) for purification and reconstitution (7). The site-directed mutagenesis was carried out by the QuikChange TM method with the primers containing mutated oligonucleotides (Table 1) and then subcloned to the expression vector pYVH6 (13,25). The mutated nucleotides were subsequently defined and confirmed by DNA sequencing.…”

“…The mutated nucleotides were subsequently defined and confirmed by DNA sequencing. The pYVH6 and its relative variants were transformed into the yeast host cell S. cerevisiae strain BJ2168 (MATa, prc-407, prb1-1122, pep4-3, leu2, trp1, ura3, GAL) and cultured according to previous methods (7,12,13). The yeast microsomal membranes enriched in H ϩ -PPases were prepared followed by solubilization and purification with Ni 2ϩ -nitrilotriacetic acid beads as described previously (7,12,13,25).…”

“…Furthermore, the dimeric structure of H ϩ -PPase was visualized by atomic force microscopy (AFM) and electron microscopy, respectively (10,11). Previous studies showed the structure-function relationship of H ϩ -PPase and identified the pivotal domains and residues involved in enzymatic activities and structural integrity (1,(12)(13)(14)(15)(16).…”

Squeezing at Entrance of Proton Transport Pathway in Proton-translocating Pyrophosphatase upon Substrate Binding

“…The core TMs encompass hydrophilic regions accommodating the catalytic site facing the cytosolic side and hydrophobic portions embedding the proton transport pathway through the membrane (14). Acidic motifs, PP i binding motif, and several highly conserved charged residues compose the major section of the catalytic site and play significant roles in PP i hydrolysis reaction (11,15,16). Further trypsinolysis analysis and structural mapping for the catalytic sites as determined by single molecule FRET (smFRET) technique demonstrated that H ϩ -PPase exists at least in three major conformations during the catalytic cycle (14,17).…”