2024-03-28T23:05:06Z
https://nagoya.repo.nii.ac.jp/oai
oai:nagoya.repo.nii.ac.jp:00005108
2023-01-16T03:53:12Z
643:666:667
Oligomerization of H+-pyrophosphatase and its structural and functional consequences
Mimura, Hisatoshi
Nakanishi, Yoichi
Maeshima, Masayoshi
前島, 正義
open access
Disulfide bond
H+-pyrophosphatase
Oligomerization
Proton pump
The H+-pyrophosphatase (H+-PPase) consists of a single polypeptide, containing 16 or 17 transmembrane domains. To determine the higher order oligomeric state of Streptomyces coelicolor H+-PPase, we constructed a series of cysteine substitution mutants and expressed them in Escherichia coli. Firstly, we analyzed the formation of disulfide bonds, promoted by copper, in mutants with single cysteine substitutions. 28 of 39 mutants formed disulfide bonds, including S545C, a substitution at the periplasmic side. The formation of intermolecular disulfide bonds suppressed the enzyme activity of several, where the substituted residues were located in the cytosol. Creating disulfide links in the cytosol may interfere with the enzyme’s catalytic function. Secondly, we prepared double mutants by introducing second cysteine substitutions into the S545C mutant. These double-cysteine mutants produced cross-linked complexes, estimated to be at least tetramers and possibly hexamers. Thirdly, we co-expressed epitope-tagged, wild type and inactive mutant H+-PPases in E. coli and confirmed the formation of oligomers by co-purifying one subunit using the epitope tag used to label the other. The enzyme activity of these oligomers was markedly suppressed. We propose that H+-PPase is present as an oligomer made up of at least two or three sets of dimers.
Elsevier
2005-07
eng
journal article
VoR
http://hdl.handle.net/2237/6661
https://nagoya.repo.nii.ac.jp/records/5108
Biochimica et Biophysica Acta
1708
3
393
403
https://nagoya.repo.nii.ac.jp/record/5108/files/2005-BBA-ScPP.pdf
application/pdf
918.3 kB
2018-02-19