Characterization of a heat-stable NADP-dependent isocitrate dehydrogenase from the obligate thermophile Thermoleophilum minutum YS-4 |
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Authors: | James F. Novotny Jr. Jerome J. Perry |
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Affiliation: | (1) Department of Microbiology, North Carolina State University, 27695 Raleigh, NC, USA |
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Abstract: | Summary A thermostable NADP-dependent isocitrite dehydrogenase (IDH; EC. 1.1.1.42) was purified from the obligately thermophilic hydrocarbonoclastic bacterium Thermoleophilum minutum YS-4 (ATCC 35265). This was accomplished by affinity chromatography and electroelution from a nondenaturing polyacrylamide gel. The enzyme has an Mr of 60 000 and is composed of two identical subunits of Mr 30 500. The amino acid composition has an Arg/Lys ratio of 4:1 and very high levels of glycine. Under nondenaturing conditions, the enzyme has a distinct difference in electrophoretic mobility relative to IDHs obtained from other genera including the genus Thermus. The secondary strcuture consists of 16% -helix, 20% -sheet, 25% -turn and 37% random coil as determined by circular dichroism spectroscopy. The optimum pH and temperature for activity were 7.2 and 75° C respectively and the apparent Kmvalues for DL-isocitrate adn NADP+ were 33 M, and 48 M, respectively. The enzyme requires divalent cations, such as Mn2+ or Mg2+ for activity. NAD+ cannot substitute for NADP+. Oxaloacetate plus glyoxylate exert considerable inhibition on IDH activity while other glycolytic and tricarboxylic acid cycle intermediates have a lesser effect. p-Chloromercuribenzoic acid was inhibitory to the IDH although isocitrate and Mn2+ offered some protection from this inactivation. The enzyme is thermostable, retaining 84% and 57% of initial activity after incubation for 1 h at 60° and 70° C, respectively. Isocitrate provided protection from thermal inactivation allowing the IDH to maintain 21% activity after 1 h at 80° C.Offprint requests to: J. J. Perry |
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