Thermal stability and biochemical properties of isocitrate dehydrogenase from the thermoacidophilic archaeon Thermoplasma acidophilum.
Isocitrate dehydrogenase [IDH; EC 1.1.1.42] from the thermoacidophilic archaeon Thermoplasma acidophilum (TaIDH) showed high thermal stability with an apparent melting temperature, T(m), of 82.2 and 84.5 degrees C at pH 7.5 and 5.8, respectively. Based on structural alignment of TaIDH with IDH from Aeropyrum pernix (ApIDH) and Archaeoglobus fulgidus (AfIDH) residues forming an aromatic cluster in the clasp-domain thought to strengthen the dimer interface in ApIDH and AfIDH were identified in the former enzyme. Moreover, TaIDH had a shortened N-terminus that may protect the enzyme from thermal denaturation. The enzyme activity of TaIDH was highest at 70 degrees C. The pH-activity profile was bell-shaped with an optimum shifted to a lower pH compared to AfIDH. The activity of TaIDH was influenced by changes in pH with a three-fold reduction in activity when the pH was shifted from the pH-optimum at 7.5 to pH 5.8. However, the specific activity at pH 5.8 was still high when compared with AfIDH. The reduction in activity at pH 5.8 was not due to instability of the enzyme as the T(m) of TaIDH was higher at pH 5.8 than at 7.5 and the enzyme retained 91% of its activity after incubation at 1 h at pH 5 and 60 degrees C. The difference in the pH-profile of TaIDH in comparison with AfIDH may thus be related to the pK(a)s of their catalytic residues involved in the initial proton abstraction and the final proton donation during the catalysis of oxidative decarboxylation of isocitrate to 2-oxoglutarate and reduced coenzyme.
Stokke
Runar
R
Department of Biology, University of Bergen, Jahnebakken 5, P.O. Box 7800, 5020, Bergen, Norway.
Birkeland
Nils-Kåre
NK
Steen
Ida Helene
IH
D019843:Archaeal Proteins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D002384:Catalysis; D004795:Enzyme Stability / Q000235:genetics; D006358:Hot Temperature; D006863:Hydrogen-Ion Concentration; D007521:Isocitrate Dehydrogenase / Q000737:chemistry* / Q000235:genetics; D007523:Isocitrates / Q000378:metabolism; D007656:Ketoglutaric Acids / Q000378:metabolism; D013822:Thermoplasma / Q000201:enzymology* / Q000235:genetics
D019843:Archaeal Proteins; D007523:Isocitrates; D007656:Ketoglutaric Acids; C034219:isocitric acid; D007521:Isocitrate Dehydrogenase