Distance variations between active sites of H(+)-pyrophosphatase determined by fluorescence resonance energy transfer.
Homodimeric H(+)-pyrophosphatase (H(+)-PPase; EC 3.6.1.1) is a unique enzyme playing a pivotal physiological role in pH homeostasis of organisms. This novel H(+)-PPase supplies energy at the expense of hydrolyzing metabolic byproduct, pyrophosphate (PP(i)), for H(+) translocation across membrane. The functional unit for the translocation is considered to be a homodimer. Its putative active site on each subunit consists of PP(i) binding motif, Acidic I and II motifs, and several essential residues. In this investigation structural mapping of these vital regions was primarily determined utilizing single molecule fluorescence resonance energy transfer. Distances between two C termini and also two N termini on homodimeric subunits of H(+)-PPase are 49.3 + or - 4.0 and 67.2 + or - 5.7 A, respectively. Furthermore, putative PP(i) binding motifs on individual subunits are found to be relatively far away from each other (70.8 + or - 4.8 A), whereas binding of potassium and substrate analogue led them to closer proximity. Moreover, substrate analogue but not potassium elicits significant distance variations between two Acidic I motifs and two His-622 residues on homodimeric subunits. Taken together, this study provides the first quantitative measurements of distances between various essential motifs, residues, and putative active sites on homodimeric subunits of H(+)-PPase. A working model is accordingly proposed elucidating the distance variations of dimeric H(+)-PPase upon substrate binding.
Huang
Yun-Tzu
YT
Department of Life Science and Institute of Bioinformatics and Structural Biology, College of Life Science, National Tsing Hua University, Hsin Chu 30013, Taiwan.
Liu
Tseng-Huang
TH
Chen
Yen-Wei
YW
Lee
Chien-Hsien
CH
Chen
Hsueh-Hua
HH
Huang
Tsu-Wei
TW
Hsu
Shen-Hsing
SH
Lin
Shih-Ming
SM
Pan
Yih-Jiuan
YJ
Lee
Ching-Hung
CH
Hsu
Ian C
IC
Tseng
Fan-Gang
FG
Fu
Chien-Chung
CC
Pan
Rong-Long
RL
D020816:Amino Acid Motifs; D020134:Catalytic Domain; D003017:Clostridium tetani / Q000201:enzymology; D019281:Dimerization; D004926:Escherichia coli / Q000201:enzymology; D031541:Fluorescence Resonance Energy Transfer / Q000379:methods*; D043564:Inorganic Pyrophosphatase / Q000737:chemistry* / Q000502:physiology*; D008024:Ligands; D008861:Microsomes / Q000378:metabolism; D009154:Mutation; D021382:Protein Sorting Signals; D021381:Protein Transport; D011755:Pyrophosphatases / Q000737:chemistry*; D013050:Spectrometry, Fluorescence
D008024:Ligands; D021382:Protein Sorting Signals; D011755:Pyrophosphatases; D043564:Inorganic Pyrophosphatase