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

Each af two histidine residues and one tryptophan residue in thermophi lic bacterium PS-3 inorganic pyrophosphatase (PPase) was replaced by a lanine. The activities of the H125A and W143A variants decreased to on e-fifth, whereas the activity of H118A remained unaltered. CD spectra in the near-UV region indicated that the conformations of the first tw o variants changed with the substitution. In contrast with wild-type P Pase, which is hexameric beyond an enzyme concentration of 0.1 mu M in the presence of Mg2+, the H118A and H125A variants cannot be assemble d from trimers into hexamers at less than an enzyme concentration of 1 0 mu M 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 a s that of wild-type PPase. These results indicated that His-118 and Hi s-125 are important for both trimer-trimer interaction and structural integrity, whereas Trp-143 is important structurally. The trimer-trime r interaction is absolutely necessary for the thermostability of the P S-3 enzyme.