Effect of heat treatment on proper oligomeric structure formation of thermostable glutamate dehydrogenase from a hyperthermophilic archaeon.
Natural glutamate dehydrogenase (Pk-GDH) was purified from hyperthermophilic archaeon Pyrococcus sp. KOD1 to homogeneity and its activity and structure were compared with those of recombinant enzyme, which was expressed in Escherichia coli. Determination of the molecular weight of these enzymes by SDS-PAGE and gel filtration revealed that the natural enzyme was purified only as a hexameric form, whereas the recombinant enzyme was purified as both monomeric and hexameric forms. Determination of the enzymatic activities indicated that only the enzyme in a hexameric form is active. Moreover, it is noted that the specific activity of the hexameric form of the recombinant enzyme is much lower than that of the natural enzyme and that circular dichroism spectra of these enzymes are distinctly different from each other. These results suggest that the structure of the hexameric form of the recombinant enzyme with low specific activity (Type I) is different from that of the natural enzyme with high specific activity (Type II). Upon heat treatment (80 degrees C, 15 min), the Type I structure was effectively converted to Type II structure and the specific activity of the enzyme was increased by 2.6-fold. Likewise, upon heat treatment (70 degrees C for 15 min), the inactive monomeric form of the recombinant enzyme was at least partially associated with the hexameric form. These results indicate that high temperature plays an important role for proper folding and oligomerization of Pk-GDH.
Abd Rahman
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RN
Department of Synthetic Chemistry and Biological Chemistry, Graduate School of Engineering, Kyoto University, Japan.
Fujiwara
S
S
Takagi
M
M
Kanaya
S
S
Imanaka
T
T
D019843:Archaeal Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D002942:Circular Dichroism; D004789:Enzyme Activation; D005969:Glutamate Dehydrogenase / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D006358:Hot Temperature; D011108:Polymers / Q000737:chemistry / Q000378:metabolism*; D019711:Pyrococcus / Q000737:chemistry / Q000201:enzymology* / Q000235:genetics; D011994:Recombinant Proteins / Q000737:chemistry
D019843:Archaeal Proteins; D011108:Polymers; D011994:Recombinant Proteins; D005969:Glutamate Dehydrogenase