Starkova N N, Koroleva E P, Rumsh L D, Ginodman L M, Rotanova T V
Shemyakin and Ovchinnikov Institute of Bioorganic Chemistry, Russian Academy of Sciences, Moscow.
FEBS Lett. 1998 Jan 30;422(2):218-20. doi: 10.1016/s0014-5793(98)00012-x.
Conserved residues of the proteolytic domain of Escherichia coli protease Lon, putative members of the classic catalytic triad (H665, H667, D676, and D743) were identified by comparison of amino acid sequences of Lon proteases. Mutant enzymes containing substitutions D676N, D743N, H665Y, and H667Y were obtained by site-directed mutagenesis. The mutant D743N retained the adenosine triphosphate (ATP)-dependent proteolytic activity, thereby indicating that D743 does not belong to the catalytic site. Simultaneously, the mutants D676N, H665Y, and H667Y lost the capacity for hydrolysis of protein substrates. The ATPase activity of these three mutants was decreased by more than an order of magnitude, which suggests a close spatial location of the ATPase and proteolytic active sites and their tight interaction in the process of protein degradation.
通过比较Lon蛋白酶的氨基酸序列，鉴定出大肠杆菌蛋白酶Lon蛋白水解结构域的保守残基，即经典催化三联体的假定成员（H665、H667、D676和D743）。通过定点诱变获得了含有D676N、D743N、H665Y和H667Y取代的突变酶。突变体D743N保留了三磷酸腺苷（ATP）依赖性蛋白水解活性，因此表明D743不属于催化位点。同时，突变体D676N、H665Y和H667Y失去了水解蛋白质底物的能力。这三个突变体的ATP酶活性降低了一个多数量级，这表明ATP酶和蛋白水解活性位点在空间上位置接近，并且在蛋白质降解过程中它们紧密相互作用。
