Inactivation and unfolding of the hyperthermophilic inorganic pyrophosphatase from Thermus thermophilus by sodium dodecyl sulfate.
Inorganic pyrophosphatase (PPase, EC 3.6.1.1) is an essential constitutive enzyme for energy metabolism and clearance of excess pyrophosphate. In this research, we investigated the sodium dodecyl sulfate (SDS)-induced inactivation and unfolding of PPase from Thermus thermophilus (T-PPase), a hyperthermophilic enzyme. The results indicated that like many other mesophilic enzymes, T-PPase could be fully inactivated at a low SDS concentration of 2 mM. Using an enzyme activity assay, SDS was shown to act as a mixed type reversible inhibitor, suggesting T-PPase contained specific SDS binding sites. At high SDS concentrations, T-PPase was denatured via a two-state process without the accumulation of any intermediate, as revealed by far-UV CD and intrinsic fluorescence. A comparison of the inactivation and unfolding data suggested that the inhibition might be caused by the specific binding of the SDS molecules to the enzyme, while the unfolding might be caused by the cooperative non-specific binding of SDS to T-PPase. The possible molecular mechanisms underlying the mixed type inhibition by SDS was proposed to be caused by the local conformational changes or altered charge distributions.
Mu
Hang
H
State Key Laboratory of Biomembrane and Membrane Biotechnology, Department of Biological Sciences and Biotechnology, Tsinghua University, Beijing 100084, China.|Yangtze Delta Region Institute of Tsinghua University, Jiaxing 314006, China.
Zhou
Sheng-Mei
SM
College of Biology and Chemical Engineering, Jiaxing University, Jiaxing 314001, China.
Xia
Yong
Y
Yangtze Delta Region Institute of Tsinghua University, Jiaxing 314006, China.
Zou
Hechang
H
Yangtze Delta Region Institute of Tsinghua University, Jiaxing 314006, China.
Meng
Fanguo
F
Yangtze Delta Region Institute of Tsinghua University, Jiaxing 314006, China.
Yan
Yong-Bin
YB
State Key Laboratory of Biomembrane and Membrane Biotechnology, Department of Biological Sciences and Biotechnology, Tsinghua University, Beijing 100084, China.
D002942:Circular Dichroism; D043564:Inorganic Pyrophosphatase / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D058767:Protein Unfolding; D011994:Recombinant Proteins / Q000096:biosynthesis / Q000737:chemistry / Q000235:genetics; D012967:Sodium Dodecyl Sulfate / Q000737:chemistry*; D013056:Spectrophotometry, Ultraviolet; D016963:Thermus thermophilus / Q000201:enzymology*
D011994:Recombinant Proteins; D012967:Sodium Dodecyl Sulfate; D043564:Inorganic Pyrophosphatase