1887

Abstract

To elucidate determinants of differences in thermostability between mesophilic and psychrophilic monomeric isocitrate dehydrogenases (IDHs) from (IDH) and (IDH), respectively, chimeric enzymes derived from the two IDHs were constructed based on the recently resolved three-dimensional structure of IDH, and several characteristics of the two wild-type and six chimeric IDHs were examined. These characteristics were then compared with those of dimeric IDH from (IDH). All recombinant enzymes with a (His)-tag attached to the N-terminal were overexpressed in the cells and purified by Ni-affinity chromatography. The catalytic activity ( ) and catalytic efficiency ( / ) of the wild-type IDH and IDH were higher than those of IDH, implying that an improved catalytic rate more than compensates for the loss of a catalytic site in the former two IDHs due to monomerization. Analyses of the thermostability and kinetic parameters of the chimeric enzymes indicated that region 2, corresponding to domain II, and particularly region 3 located in the C-terminal part of domain I, are involved in the thermolability of IDH, and that the corresponding two regions of IDH are important for exhibiting higher catalytic activity and affinity for isocitrate than IDH. The relationships between the stability, catalytic activity and structural characteristics of IDH and IDH are discussed.

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2005-04-01
2024-10-05
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