PPase on Substitution of Ser-89 with Carboxylic Amino Acids1

Serine 89 of the inorganic pyrophosphatase (PPase) subunit from thennophilic bacte-rium PS-3 (PS-3) was replaced with glycine, alanine, threonine, glutamic acid, or aspar-tic 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 5 mM MgCl ̂ at 70*C for 1 h, 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 1 h incubation. The wild-type PPase, S89D and S89E were each a hexamer before and after incubation at 80"C for 1 h, while S89G and S89A comprised a mixture of a hexamer and a trimer both before and after incubation at 80"C for 1 h. 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 th