Abstract
H(+)-pyrophosphatase (H(+)-PPase) catalyzes pyrophosphate-driven proton transport against the electrochemical potential gradient in various biological membranes. All 50 of the known H(+)-PPase amino acid sequences contain four invariant glutamate residues. In this study, we use site-directed mutagenesis in conjunction with functional studies to determine the roles of the glutamate residues Glu(197), Glu(202), Glu(550), and Glu(649) in the H(+)-PPase of Rhodospirillum rubrum (R-PPase). All residues were replaced with Asp and Ala. The resulting eight variant R-PPases were expressed in Escherichia coli and isolated as inner membrane vesicles. All substitutions, except E202A, generated enzymes capable of PP(i) hydrolysis and PP(i)-energized proton translocation, indicating that the negative charge of Glu(202) is essential for R-PPase function. The hydrolytic activities of all other PPase variants were impaired at low Mg(2+) concentrations but were only slightly affected at high Mg(2+) concentrations, signifying that catalysis proceeds through a three-metal pathway in contrast to wild-type R-PPase, which employs both two- and three-metal pathways. Substitution of Glu(197), Glu(202), and Glu(649) resulted in decreased binding affinity for the substrate analogues aminomethylenediphosphonate and methylenediphosphonate, indicating that these residues are involved in substrate binding as ligands for bridging metal ions. Following the substitutions of Glu(550) and Glu(649), R-PPase was more susceptible to inactivation by the sulfhydryl reagent mersalyl, highlighting a role of these residues in maintaining enzyme tertiary structure. None of the substitutions affected the coupling of PP(i) hydrolysis to proton transport.
Highlights
Proton pumping pyrophosphatase (Hϩ-PPase)1 is an integral membrane protein that utilizes the energy released upon the hydrolysis of PPi to transport protons across the membrane against the electrochemical potential gradient [1,2,3]
Hydrolytic and Proton-pumping Activities of R-PPase Variants—Each of the four conserved glutamate residues in RPPase was individually substituted with aspartate and alanine, and the resulting variants were expressed in E. coli C43(DE3)
All variants capable of PPi hydrolysis were active in PPi-energized Hϩ translocation in E. coli inner membrane vesicles (IMVs) (Fig. 2)
Summary
Hϩ-PPase, proton-translocating inorganic pyrophosphatase; R-PPase, Rhodospirillum rubrum Hϩ-PPase; AMDP, aminomethylenediphosphonate; IMV, inner membrane vesicles; MDP, methylenediphosphonate; CAPS, 3-(cyclohexylamino)propanesulfonic acid; CAPSO, 3-(cyclohexylamino)-2-hydroxyl-1-propanesulfonic acid; HEPPSO, N-2-hydroxyethylpiperazine-NЈ-2-hydroxypropanesulfonic acid; MES, 2-(N-morpholino)ethanesulfonic acid; MOPS, 3-(N-. Sequences from different species display at least 30% identity, and most of the 60 conserved residues are clustered in three hydrophilic regions comprising cytosolic loops that probably form the active site These residues include four Glu residues (Fig. 1) that are present in all 50 of the known Hϩ-PPase sequences. The development of expression systems for plant Hϩ-PPases in yeast Saccharomyces cerevisiae [9] and bacterial Hϩ-PPases in Escherichia coli [18] has paved the way for site-directed mutagenesis of these proton pumps This approach was successfully employed to identify membrane-embedded charged residues contributing to dicyclohexylcarbodiimide binding in Hϩ-PPases [19], reactive Cys residues [18, 20], and those residues that determine the potassium requirement of Hϩ-PPases [16]. Our data indicate that Glu197 and Glu202 contribute to substrate binding, whereas Glu550 and Glu649 control R-PPase conformation
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