Metal ions, isocitrate dehydrogenase

Isocitrate dehydrogenases have been studied in extreme thermophiles mainly from the point of view of elucidation of mechanisms of thermostability. This has included the studies of Hibino et al. [32] on the enzyme from a Bacillus stearothermophilus strain grown at 65 °C. In the presence of substrate the enzyme was markedly stabilized as judged by retention of tertiary conformation, reflected by the dramatically improved resistance to denaturation by urea. The thermostability of the partially purified isodtrate dehydrogenase from Thermus aquaticus was shown to be dependent upon the buffer used and on the addition of isodtrate [135]. This enzyme has also been purified from Thermus thermophilus HB8 [136]. The enzyme had a dimeric structure with identical 57.5 kDa subunits as judged by SDS-polyacrylamide gel electrophoresis, whilst gel filtration of the native enzyme on two different matrices gave values of 95 kDa and 120 kDa. The enzyme had a pH optimum of 7.8, required a divalent metal ion for activity (Mn " > Mg " ) and was NADP dependent, although NAD" was shown to replace NADP" with low efficiency. 

More reeently, Mesecar and Koshland [32], following an investigation of the enzyme isoeitrate dehydrogenase, have found that the three-point model does not always hold. Examination of metal-free crystals of the enzyme strueture reveals that only the 25,37 -(L)-isocitrate binds, whereas in the presenee of magnesium ions only the 22 ,3iS -(D)-enantiomer binds. Examination of x-ray structures of the two enzyme-substrate complexes reveals three common binding sites for both enantiomeric substrates that differ at a fourth site. Based on their observations, the authors proposed that the three-point model is only applicable if the assumption is made that the substrate can approach a planar surface from one direction. Thus a fourth location, either a direction requirement or an additional binding site, is essential to distinguish between a pair of enantiomers (Fig. 5). 

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