Studies on NADP⁺-specific Isocitrate Dehydrogenase from an Extreme Thermophile, Thermus flavus AT-62

Partial Purification and Properties

Abstract

1. NADP⁺-specific isocitrate dehydrogenase [EC 1.1.1.42] was partially purified by about 440-fold from an extreme thermophile, Thermus flavus AT-62.
2. Remarkable thermostability of the enzyme was confirmed. The enzyme was not inactivated after 60 min at 70°, and the activity was lost only slowly at 80°. Above 90°, however, rapid inactivation was observed.
3. The dehydrogenase was susceptible to concerted inhibition by oxaloacetate plus glyoxylate. In the presence of oxaloacetate plus glyoxylate (each 1mM), 75% inhibi-tion was observed.
4. The degree of inhibition of the enzyme by oxaloacetate plus glyoxylate decreased markedly above 60°. The affinity of the enzyme for isocitrate and NADP⁺ was also reduced markedly above 60°. The activation energy calculated from Arrhenius plots below and above 60° were 14, 500 and 8, 000 cal per mole, respectively.
These observations suggest a possible conformation change of the enzyme protein at a transition temperature of 60°, and the physiological significance of this in the adaptation of thermophiles to elevated temperatures is discussed.