Deletion of Ala144-Lys145 in Thermus thermophilus inorganic pyrophosphatase suppresses thermal aggregation.
The regions contributing to the thermostability of inorganic pyrophosphatase (PPase, EC 3.6.1.1) from Thermus thermophilus (Tth) were deduced in our previous study by random chimeragenesis, one of them being estimated to be Ala144-Lys145 [Satoh, T., Takahashi, Y., Oshida, N., Shimizu, A., Shinoda, H., Watanabe, M., and Samejima, T. (1999) Biochemistry 38, 1531-1536]. Therefore, we investigated the contributions of these two residues in Tth by preparing a deletion mutant (del.144-145 mutant) of Tth PPase. We examined its thermostability in terms of the CD and fluorescence spectra, and the thermal change in the enzymatic activity. The thermostability of the enzymatic activity of the del.144-145 mutant was similar to that of the wild type Tth PPase, 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 mutant after heating at 90 degrees C was higher than that of the unheated one, whereas the wild type aggregated under the same conditions. Therefore, we can conclude that although the Ala144-Lys145 residues in Tth PPase may partly cause thermal aggregation, the deletion of these residues may stabilize the Tth PPase molecule structurally against heating and suppress thermal aggregation.
Satoh
T
T
Department of Chemistry, College of Science and Engineering, Aoyama Gakuin University, Tokyo, 157-8572, Japan.
Oshida
N
N
Ono
M
M
Hattori
M
M
Ohta
T
T
Watanabe
M
M
Shinoda
H
H
Takahashi
Y
Y
Lee
J S
JS
Samejima
T
T
D000409:Alanine / Q000737:chemistry*; D001483:Base Sequence; D002942:Circular Dichroism; D017931:DNA Primers; D004795:Enzyme Stability; D006358:Hot Temperature; D043564:Inorganic Pyrophosphatase; D008239:Lysine / Q000737:chemistry*; D011487:Protein Conformation; D011755:Pyrophosphatases / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D011993:Recombinant Fusion Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D017384:Sequence Deletion; D013050:Spectrometry, Fluorescence; D016963:Thermus thermophilus / Q000201:enzymology*
D017931:DNA Primers; D011993:Recombinant Fusion Proteins; D011755:Pyrophosphatases; D043564:Inorganic Pyrophosphatase; D008239:Lysine; D000409:Alanine