A highly stable NADP-dependent isocitrate dehydrogenase from Thermus thermophilus HB8: purification and general properties.
H Eguchi, T Wakagi, T Oshima
Index: Biochim. Biophys. Acta 990 , 133, (1989)
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Abstract
NADP-dependent isocitrate dehydrogenase (EC 1.1.1.42) was purified to electrophoretic homogeneity from an extremely thermophilic bacterium, Thermus thermophilus HB8, and shown to be a dimeric protein of molecular weight 115,000, with a pI of 5.5. The amino acid composition of the present enzyme was similar to that reported for other bacterial counterparts, except for a high Arg/Lys ratio and a low Cys content. Divalent cations, such as Mn2+ and Mg2+, were essential for activity. The optimal pH was 7.8 at 55 degrees C. The Km values for NADP and D-isocitrate were 6.3 and 8.8 microM, respectively, with a Vmax of 77.6 mumol/min per mg at 55 degrees C. NAD was able to replace NADP with low efficiency. Backward reaction at 40 degrees C indicated that the Km value for 2-oxoglutarate was 63 microM with a Vmax of 4% that of the forward reaction at that temperature. The enzyme was highly stable against high temperature and denaturing reagents.