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Essential amino acid residues in the central transmembrane domains and loops for energy coupling of Streptomyces coelicolor A3(2) H+-pyrophosphatase
- Source :
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BBA - Bioenergetics . Jul2007, Vol. 1767 Issue 7, p930-939. 10p. - Publication Year :
- 2007
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Abstract
- Abstract: The H+-translocating inorganic pyrophosphatase is a proton pump that hydrolyzes inorganic pyrophosphate. It consists of a single polypeptide with 14−17 transmembrane domains, and is found in a range of organisms. We focused on the second quarter region of Streptomyces coelicolor A3(2) H+-pyrophosphatase, which contains long conserved cytoplasmic loops. We prepared a library of 1536 mutants that were assayed for pyrophosphate hydrolysis and proton translocation. Mutant enzymes with low substrate hydrolysis and proton-pump activities were selected and their DNAs sequenced. Of these, 34 were single-residue substitution mutants. We generated 29 site-directed mutant enzymes and assayed their activity. The mutation of 10 residues in the fifth transmembrane domain resulted in low coupling efficiencies, and a mutation of Gly198 showed neither hydrolysis nor pumping activity. Four residues in cytoplasmic loop e were essential for substrate hydrolysis and efficient H+ translocation. Pro189, Asp281, and Val351 in the periplasmic loops were critical for enzyme function. Mutation of Ala357 in periplasmic loop h caused a selective reduction of proton-pump activity. These low-efficiency mutants reflect dysfunction of the energy-conversion and/or proton-translocation activities of H+-pyrophosphatase. Four critical residues were also found in transmembrane domain 6, three in transmembrane domain 7, and five in transmembrane domains 8 and 9. These results suggest that transmembrane domain 5 is involved in enzyme function, and that energy coupling is affected by several residues in the transmembrane domains, as well as in the cytoplasmic and periplasmic loops. H+-pyrophosphatase activity might involve dynamic linkage between the hydrophilic and transmembrane domains. [Copyright &y& Elsevier]
- Subjects :
- *AMINO acids
*PROTON pump inhibitors
*STREPTOMYCES
*HYDROLYSIS
Subjects
Details
- Language :
- English
- ISSN :
- 00052728
- Volume :
- 1767
- Issue :
- 7
- Database :
- Academic Search Index
- Journal :
- BBA - Bioenergetics
- Publication Type :
- Academic Journal
- Accession number :
- 25750411
- Full Text :
- https://doi.org/10.1016/j.bbabio.2007.03.014