Dissection of functional domains of the yeast proton-pumping ATPase by directed mutagenesis.

Evidence for Two Catalytic Sites in the Functional Unit of H+-ATPase from Higher Plants

“…The number of phosphorylating sites per monomer is unknown in this enzyme but from the amino acid sequence the existence of more than one is improbable (Cid et al, 1987;Portillo and Serrano, 1988;Serrano, 1988b). It could be conceived that the H+-ATPase functional unit is a dimer with one ATP-binding site per monomer.…”

Altered plasma membrane H+‐ATPase from the Dio‐9‐resistant pmal‐2 mutant of Schizosaccharomyces pombe

“…JV2Y and JV37 that results in amino acid substitution of Lys250 (AAG codon) by threonine (ACG codon). This residue, located close to Gly268 within thc phosphatase domain [9] or the transduction domain, also called the P-stranded sector [lo], is conserved (region d, Fig. 1) in the pmal and pma2 H+-ATPascs of S. pomhe [7,211 and S. cerevisiae [4, 221, in the pmal H + -ATPase of N .…”

“…The closely related polypeptides are folded similarly in the plasma membrane, with 8 -12 putative transmembrane segments delimiting two major cytoplasmic regions. The largest hydrophilic region contains the nucleotide-binding and phosphorylation domains [9]. A smaller hydrophilic domain, rcferred to as an energy-transduction or P-stranded [lo] domain, is believed to be essential for protein phosphatase activity [9] and/or for conformational changes at the level of the phosphoenzyme [I 1 I.…”

“…The largest hydrophilic region contains the nucleotide-binding and phosphorylation domains [9]. A smaller hydrophilic domain, rcferred to as an energy-transduction or P-stranded [lo] domain, is believed to be essential for protein phosphatase activity [9] and/or for conformational changes at the level of the phosphoenzyme [I 1 I. The pmal-1 mutation, affecting the fission yeast H +-ATPase, was originally selected on the basis of Dio-9 resistance [12] and was later shown to result from rcplacemcnt of Gly268 by an aspartate [7].…”

Localization of a putative magnesium‐binding site within the cytoplasmic domain of the sarcoplasmic reticulum Ca2+‐ATPase

“…One of these regions, located around residues 696-707 in the CaZ+-ATPase, the socalled 'hinge' region, presents the highest degree of conservation among the sequences of P-type ATPases (Serrano, 1988) It has been shown that mutations of Thr701, Gly702, Asp703 or Asp707 strongly reduce or totally inhibit the calcium-transport activity of the Ca'+-ATPase (Clarke et al, 1990). The corresponding sequence in NdK-ATPase can be labeled by 5'-(p-fluorosulfonyl)benzoyladenosine, an ATP analogue that inhibits the ATPase activity of this enzyme (Ohta et al, 1986).These observations indicate that the integrity of this part of the protein is essential for the activity of the P-type AT- Pases, and suggest that it may be involved in the binding of ATP to the active site.It has been proposed that the area surrounding the Asp638 of a yeast proton-pumping ATPase (Serrano, 1988;Portillo and Serrano, 1988) could be involved in the binding of the y phosphate of ATP through a chelated magnesium ion. We found that this proposal is supported by the fact that the corresponding region (Glu696-Asp707) in the Ca2--ATPase shows significant similarity to EF-hand divalent-cation-binding structures.…”