2001 年 129 巻 6 号 p. 955-961
Serine 89 of the inorganic pyrophosphatase (PPase) subunit from thermophilic bacterium PS-3 (PS-3) was replaced with glycine, alanine, threonine, glutamic acid, or aspartic acid by the PCR-mutagenesis method with Mut-1 in order to determine the contribution of this serine residue to the thermostability and structural integrity of the enzyme molecule. S89G, S89A, and S89T showed reduced catalytic activity, whereas S89D and S89E showed increased enzyme activity. S89G, S89A, and S89T as well as the wild-type PPase were stable in the presence of 5mM MgCl2 at 70°C for 1h, but were inactivated rapidly with time at 80°C. On the contrary, S89D and S89E were stable at 80°C, showing more than 95% of the original activity after 1h incubation. The wild-type PPase, S89D and S89E were each a hexamer before and after incubation at 80°C for 1h, while S89G and S89A comprised a mixture of a hexamer and a trimer both before and after incubation at 80°C for 1h. On the other hand, S89T was a mixture of a hexamer, a trimer and a monomer, and it was partially precipitated during heat treatment at 80°C. The CD spectra of the recombinant enzymes in the far-ultraviolet region were the same as that of the wild-type PPase, whereas those of S89G, S89A, and S89T as well as the wild-type PPase were markedly different after heat treatment, although those of S89D and S89E did not change. The present study suggested that local small change(s) in the network of interactions among amino acid residues on replacement at position 89 led to the PS-3 PPase molecule being unable to form a hexamer from trimers or to dissociate into monomers in some cases without a significant change in the backbone conformation. It was also suggested that the partial disordering of the conformation of PS-3 PPase caused by heat depended on the degree of hydrophilicity in the vicinity of position 89.
