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Identification of essential lysines involved in substrate binding of vacuolar H+-pyrophosphatase.


ABSTRACT: H+-translocating pyrophosphatase (H+-PPase; EC 3.6.1.1) drives proton transport against an electrochemical potential gradient by hydrolyzing pyrophosphate (PPi) and is found in various endomembranes of higher plants, bacteria, and some protists. H+-PPase contains seven highly conserved lysines. We examined the functional roles of these lysines, which are, for the most part, found in the cytosolic regions of mung bean H+-PPase by site-directed mutagenesis. Construction of mutants that each had a cytosolic and highly conserved lysine substituted with an alanine resulted in dramatic drops in the PPi hydrolytic activity. The effects caused by ions on the activities of WT and mutant H+-PPases suggest that Lys-730 may be in close proximity to the Mg2+-binding site, and the great resistance of the K694A and K695A mutants to fluoride inhibition suggests that these lysines are present in the active site. The modifier fluorescein 5'-isothiocyanate (FITC) labeled a lysine at the H+-PPase active site but did not inhibit the hydrolytic activities of K250A, K250N, K250T, and K250S, which suggested that Lys-250 is essential for substrate binding and may be involved in proton translocation. Analysis of tryptic digests indicated that Lys-711 and Lys-717 help maintain the conformation of the active site. Proteolytic evidence also demonstrated that Lys-250 is the primary target of trypsin and confirmed its crucial role in H+-PPase hydrolysis.

SUBMITTER: Lee CH 

PROVIDER: S-EPMC3069399 | biostudies-literature | 2011 Apr

REPOSITORIES: biostudies-literature

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Identification of essential lysines involved in substrate binding of vacuolar H+-pyrophosphatase.

Lee Chien-Hsien CH   Pan Yih-Jiuan YJ   Huang Yun-Tzu YT   Liu Tseng-Huang TH   Hsu Shen-Hsing SH   Lee Ching-Hung CH   Chen Yen-Wei YW   Lin Shih-Ming SM   Huang Lin-Kun LK   Pan Rong-Long RL  

The Journal of biological chemistry 20110203 14


H+-translocating pyrophosphatase (H+-PPase; EC 3.6.1.1) drives proton transport against an electrochemical potential gradient by hydrolyzing pyrophosphate (PPi) and is found in various endomembranes of higher plants, bacteria, and some protists. H+-PPase contains seven highly conserved lysines. We examined the functional roles of these lysines, which are, for the most part, found in the cytosolic regions of mung bean H+-PPase by site-directed mutagenesis. Construction of mutants that each had a  ...[more]

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