Each of two histidine residues and one tryptophan residue in thermophilic bacterium PS-3 inorganic pyrophosphatase (PPase) was replaced by alanine. The activities of the H125A and W143A variants decreased to one-fifth, whereas the activity of H118A remained unaltered. CD spectra in the near-UV region indicated that the conformations of the first two variants changed with the substitution. In contrast with wild-type PPase, which is hexameric beyond an enzyme concentration of 0.1 µM in the presence of Mg2+, the H118A and H125A variants cannot be assembled from trimers into hexamers at less than an enzyme concentration of 10 µM even at a higher concentration of Mg2+. In particular, H118A was irreversibly inactivated in a diluted state. In contrast, the enzyme concentration dependence of W143A PPase activity was almost the same as that of wild-type PPase. These results indicated that His-118 and His-125 are important for both trimer–trimer interaction and structural integrity, whereas Trp-143 is important structurally. The trimer–trimer interaction is absolutely necessary for the thermostability of the PS-3 enzyme.
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Research Article|
April 01 1998
Effect of replacement of His-118, His-125 and Trp-143 by alanine on the catalytic activity and subunit assembly of inorganic pyrophosphatase from thermophilic bacterium PS-3
Motoko AOKI;
Motoko AOKI
*Institute of High Polymer Research, Faculty of Textile Science and Technology, Shinshu University, 3-15-1, Tokida, Ueda, Nagano 386, Japan
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Toshio UCHIUMI;
Toshio UCHIUMI
*Institute of High Polymer Research, Faculty of Textile Science and Technology, Shinshu University, 3-15-1, Tokida, Ueda, Nagano 386, Japan
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Eiichi TSUJI;
Eiichi TSUJI
†Central Research Laboratory, Kissei Pharmaceutical Co., 3465-1 Kashiwabara, Hotaka-machi, Minamiazumigun, Nagano 399-83, Japan
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Akira HACHIMORI
Akira HACHIMORI
1
*Institute of High Polymer Research, Faculty of Textile Science and Technology, Shinshu University, 3-15-1, Tokida, Ueda, Nagano 386, Japan
1To whom correspondence should be addressed.
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Publisher: Portland Press Ltd
Received:
September 16 1997
Revision Received:
December 01 1997
Accepted:
December 12 1997
Online ISSN: 1470-8728
Print ISSN: 0264-6021
The Biochemical Society, London © 1998
1998
Biochem J (1998) 331 (1): 143–148.
Article history
Received:
September 16 1997
Revision Received:
December 01 1997
Accepted:
December 12 1997
Citation
Motoko AOKI, Toshio UCHIUMI, Eiichi TSUJI, Akira HACHIMORI; Effect of replacement of His-118, His-125 and Trp-143 by alanine on the catalytic activity and subunit assembly of inorganic pyrophosphatase from thermophilic bacterium PS-3. Biochem J 1 April 1998; 331 (1): 143–148. doi: https://doi.org/10.1042/bj3310143
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