Effects of replacement of prolines with alanines on the catalytic activity and thermostability of inorganic pyrophosphatase from thermophilic bacterium PS-3.
Each of the 10 proline residues of the inorganic pyrophosphatase (PPase) subunit of thermophilic bacterium PS-3 (PS-3) was replaced with alanine by the PCR-mutagenesis method. The variants were classified into three groups according to the effects of the replacements on their catalytic activities in 20 mM Tris-HCl, pH 7.8, containing 5 mM MgCl(2): the catalytic activity was (i) slightly affected (P39A and P69A), (ii) considerably reduced (P14A, P43A, P59A, and P116A), and (iii) completely or almost completely abolished (P72A, P100A, P104A, and P146A). HPLC-gel chromatography in the presence of 5 mM MgCl(2) revealed the following subunit assembly of the variants: group (i), a hexamer; group (ii), a hexamer or a mixture of a hexamer and a trimer, although the hexamer was predominant; and group (iii), a trimer or a monomer. The thermostability of the variant PPases depended upon the amount of hexamer remaining in the presence of Mg(2+) at high temperature. The results indicated that the hexamer state formed through protomer-protomer and trimer-trimer interactions is necessary for the PS-3 PPase to retain the correct structure for full catalytic activity and thermostability.
Masuda
Hideki
H
Institute of High Polymer Research, Faculty of Textile Science and Technology, Shinshu University, Tokida, Ueda, Nagano 386-8567, Japan.
Uchiumi
Toshio
T
Wada
Michiko
M
Ichiba
Tetsuroh
T
Hachimori
Akira
A
D000409:Alanine / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D019943:Amino Acid Substitution; D002384:Catalysis; D002942:Circular Dichroism; D004789:Enzyme Activation; D004795:Enzyme Stability; D004926:Escherichia coli / Q000201:enzymology*; D006358:Hot Temperature; D043564:Inorganic Pyrophosphatase; D017354:Point Mutation; D011392:Proline / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D011755:Pyrophosphatases / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D016963:Thermus thermophilus / Q000201:enzymology*
D011392:Proline; D011755:Pyrophosphatases; D043564:Inorganic Pyrophosphatase; D000409:Alanine