Identification of essential lysines involved in substrate binding of vacuolar H+-pyrophosphatase |
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Authors: | Lee Chien-Hsien Pan Yih-Jiuan Huang Yun-Tzu Liu Tseng-Huang Hsu Shen-Hsing Lee Ching-Hung Chen Yen-Wei Lin Shih-Ming Huang Lin-Kun Pan Rong-Long |
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Institution: | Department of Life Science and Institute of Bioinformatics and Structural Biology, College of Life Science, National Tsing Hua University, Hsin Chu 30043, Taiwan. |
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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. |
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Keywords: | Chemical Modification Plant Proton Transport Site-directed Mutagenesis Vacuolar Acidification H+-Pyrophosphatase |
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