To elucidate the mechanism for the regulation of aspartate kinase (AK) via feedback inhibition, we constructed several chimeric enzymes between Bacillus subtilis AK II, a lysine-sensitive mesophilic enzyme, and Thermus flavus AK, a threonine-sensitive thermostable enzyme, each having the same alpha(2)beta(2)-type tetrameric structure. A chimeric AK, named BTT, composed of the chimeric alpha subunit that comprises of the N-terminal catalytic region From B. subtilis AK II and the C-terminal region from T.flavus, and the beta subunit from T.flavus, was inhibited only by threonine. Another chimeric enzyme, BT, which has a similar structure to that of BTT but lacks the beta subunit, having alpha(2)-type homo-dimeric structure, was also responsive only to threonine. However, the addition of threonine enhanced the activity of BT. These results indicate the regulatory function of C-terminal region and P subunit in AK. BTT showed extremely high thermostability comparable to that of T.flavus, suggesting that the P subunit also contributed to the stability of the AK. (C) 2004 Elsevier Inc. All rights reserved.