Elucidating the Role of Conserved Glutamates in H+-pyrophosphatase of Rhodospirillum rubrum

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.

Disulfide‐bond formation in the H+‐pyrophosphatase of Streptomyces coelicolor and its implications for redox control and enzyme structure

“…The motifs GGIFTKAADVGADLVGKVE (loop e ) and EDDPRN (loop m ) have been demonstrated to form the catalytic site [24,38,43]. The glutamate residues of motif EVRRQ (loop m ) play a role in maintaining the tertiary structure of the R. rubrum enzyme [43]. These observations in conjunction with the present results underline the importance of loops e and m for substrate hydrolysis.…”

“…Loops e and m are cytoplasmic [4] and contain sequences that are conserved in the H + ‐PPases of various organisms, such as GGIFTKAADVGADLVGKVE, EDDPRN and IADNVGDNVGDCA (loop e , shared residues are underlined), and EVRRQ (loop m ). The motifs GGIFTKAADVGADLVGKVE (loop e ) and EDDPRN (loop m ) have been demonstrated to form the catalytic site [24,38,43]. The glutamate residues of motif EVRRQ (loop m ) play a role in maintaining the tertiary structure of the R. rubrum enzyme [43].…”

Expression of Functional Streptomyces coelicolor H+-Pyrophosphatase and Characterization of Its Molecular Properties

“…H + -PPases are excellent models for research on the coupling between PPi hydrolysis and active H + transport, and their structure-function relationships have been investigated extensively (4,(12)(13)(14)(15)(16)(17). Studies using heterologous-expression systems to analyse mutant forms of these enzymes have revealed their membrane topology and identified several functional motifs.…”

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

“…In mung bean H ϩ -PPase, DX 7 KXE and acidic motifs accommodate some group I lysines, Lys-261 and Lys-730, respectively. Furthermore, soluble and membrane-bound H ϩ -PPases share many common catalytic characteristics and contain many similar essential amino acid residues (14,16,18,32,33). Based on the structure of the soluble PPase active site, including the two motifs and a substratebinding lysine (see Ref.…”

“…the third, the sixth, and the eighth (Fig. 1), which form an enzymatic domain (1,18,19). The conserved DX 7 KXE motif of the third cytosolic loop is also found in soluble PPase and has been implicated as a substrate catalysis/binding site (1,11).…”