Deletion of Ala144-Lys145 in Thermus thermophilus inorganic pyrophosphatase suppresses thermal aggregation

The regions contributing to the thermostability of inorganic pyrophosphatas e (PPase, EC 3.6.1.1) from Thermus thermophilus (Tth) were deduced in our p revious study by random chimeragenesis, one of them being estimated to be A la144-Lys145 [Satoh, T,, Takahashi, Y., Oshida, N., Shimizu, A., Shinoda, H ., Watanabe, M., and Samejima, T. (1999) Biochemistry 38, 1531-1536], There fore, we investigated the contributions of these two residues in nh by prep aring a deletion mutant (del.144-145 mutant) of Tth PPase. We examined its thermostability in terms of the CD and fluorescence spectra, and the therma l change in the enzymatic activity. The thermostability of the enzymatic ac tivity of the del.144-145 mutant was similar to that of the wild type Tth P Pase, whereas this mutant was more stable against heating. Furthermore, we compared the thermal aggregation of the wild type with that of the del.144- 145 mutant. We found that the thermal aggregation of the mutant was reduced relative to that of the wild type. Moreover, the molecular weight of the m utant after heating at 90 degrees C was higher than that of the unheated on e, whereas the wild type aggregated under the same conditions. Therefore, w e can conclude that although the Ala144-Lys145 residues in Tth PPase may pa rtly cause thermal aggregation, the deletion of these residues may stabiliz e the nh PPase molecule structurally against heating and suppress thermal a ggregation.