title: 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.
abstract: 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 microM 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 microM 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.
authors.affiliation: Institute of High Polymer Research, Faculty of Textile Science and Technology, Shinshu University, 3-15-1, Tokida, Ueda, Nagano 386, Japan.
authors.forename: M
authors.initials: M
authors.lastname: Aoki
authors.forename: T
authors.initials: T
authors.lastname: Uchiumi
authors.forename: E
authors.initials: E
authors.lastname: Tsuji
authors.forename: A
authors.initials: A
authors.lastname: Hachimori
mesh_terms: D000409:Alanine; D019943:Amino Acid Substitution; D004789:Enzyme Activation; D004926:Escherichia coli; D006639:Histidine; D043564:Inorganic Pyrophosphatase; D011487:Protein Conformation; D011755:Pyrophosphatases / Q000737:chemistry* / Q000378:metabolism*; D013329:Structure-Activity Relationship; D014364:Tryptophan
chemical_list: D006639:Histidine; D014364:Tryptophan; D011755:Pyrophosphatases; D043564:Inorganic Pyrophosphatase; D000409:Alanine