A highly stable NADP-dependent isocitrate dehydrogenase from Thermus thermophilus HB8: purification and general properties.
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.
Eguchi
H
H
Department of Life Science, Tokyo Institute of Technology, Kanagawa, Japan.
Wakagi
T
T
Oshima
T
T
D000596:Amino Acids / Q000032:analysis; D004591:Electrophoresis, Polyacrylamide Gel; D007521:Isocitrate Dehydrogenase / Q000302:isolation & purification* / Q000378:metabolism; D007523:Isocitrates / Q000378:metabolism; D007526:Isoelectric Point; D007700:Kinetics; D008274:Magnesium / Q000378:metabolism; D008345:Manganese / Q000378:metabolism; D008970:Molecular Weight; D009249:NADP / Q000378:metabolism*; D013824:Thermus / Q000201:enzymology*
D000596:Amino Acids; D007523:Isocitrates; D008345:Manganese; D009249:NADP; C034219:isocitric acid; D007521:Isocitrate Dehydrogenase; D008274:Magnesium