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...

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Bibliographic Details
Published in:Journal of biochemistry (Tokyo) 2002-01, Vol.131 (1), p.53-58
Main Authors: Masuda, Hideki, Uchiumi, Toshio, Wada, Michiko, Ichiba, Tetsuroh, Hachimori, Akira
Format: Article
Language:English
Subjects:
Alanine - chemistry
Alanine - genetics
Alanine - metabolism
Amino Acid Substitution
Catalysis
Circular Dichroism
Enzyme Activation
Enzyme Stability
Escherichia coli - enzymology
Hot Temperature
Inorganic Pyrophosphatase
mutagenesis
Point Mutation
polymerase chain reaction
pro-line
Proline - chemistry
Proline - genetics
Proline - metabolism
Pyrophosphatases - chemistry
Pyrophosphatases - genetics
Pyrophosphatases - metabolism
thermophilic bacterium PS-3
Thermus thermophilus - enzymology
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Summary: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 MgCLj: 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 MgCla 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 Mg2+ at high temperature. The results indicated that the hexamer state formed through pro-tomer-protomer and trimer-trimer interactions is necessary for the PS-3 PPase to retain the correct structure for full catalytic activity and thermostability
ISSN:0021-924X
DOI:10.1093/oxfordjournals.jbchem.a003077