An extremely stable inorganic pyrophosphatase purified from the cytosol of a thermoacidophilic archaebacterium, Sulfolobus acidocaldarius strain 7.
A highly active inorganic pyrophosphatase was purified to electrophoretical homogeneity from the cytosol of Sulfolobus acidocaldarius strain 7, an extremely thermoacidophilic archaebacterium. The enzyme has an apparent molecular mass of 80 kDa as estimated by gel permeation chromatography, and showed a 21-kDa polypeptide on SDS-PAGE, suggesting that the archaebacterial enzyme is similar to most of the eubacterial pyrophosphatases rather than eukaryotic ones. The pI = 5.1. The enzyme showed relatively high content of Pro and low content of Ser plus Thr. The optimal pH was 6.5 (at 56 degrees C). From the Arrhenius plot an activation energy of 11.2 kcal/mol was obtained between 37-95 degrees C. The specific activity was 617 mumol Pi release min-1 mg-1 at 56 degrees C. The S. acidocaldarius pyrophosphatase was extremely stable. Complete activity remained after incubation at 100 degrees C for 10 min. No dissociation into subunit or unfolding of polypeptide chain occurred in the presence of 8 M urea. Experiments using guanidine-HCl suggested that the transition between a native tetrameric state and an unfolded state is completely reversible, and essentially independent of any additional factors such as divalent metal cation or dithiothreitol.
Wakagi
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Department of Life Science, Faculty of Bioscience and Bioengineering, Tokyo Institute of Technology, Yokohama, Japan.
Lee
C H
CH
Oshima
T
T
D000596:Amino Acids / Q000737:chemistry; D002942:Circular Dichroism; D003600:Cytosol / Q000201:enzymology*; D011756:Diphosphates / Q000737:chemistry; D004789:Enzyme Activation / Q000187:drug effects; D004795:Enzyme Stability / Q000187:drug effects; D006863:Hydrogen-Ion Concentration; D007526:Isoelectric Point; D008970:Molecular Weight; D011755:Pyrophosphatases / Q000737:chemistry / Q000302:isolation & purification*; D013379:Substrate Specificity; D016945:Sulfolobus acidocaldarius / Q000187:drug effects / Q000201:enzymology* / Q000254:growth & development; D013696:Temperature
D000596:Amino Acids; D011756:Diphosphates; D011755:Pyrophosphatases