Tsai H J, Wilson J E
Department of Biochemistry, Michigan State University, East Lansing, Michigan, 48824-1319, USA.
Arch Biochem Biophys. 1997 Feb 15;338(2):183-92. doi: 10.1006/abbi.1996.9850.
Previous studies have shown that catalytic function is associated with both halves of the Type II isozyme of mammalian hexokinase, while the Type I isozyme is functionally differentiated into a catalytic C-terminal half and regulatory N-terminal half. The Type III isozyme has now been shown to be similar to the Type I isozyme in its functional organization. Chimeras composed of the N-terminal half of Type III hexokinase and the C-terminal half of either Type I or Type II hexokinase have activities that can be attributed to the C-terminal half and are similar in activity to chimeras composed of the C-terminal half of Type III and the intrinsically inactive N-terminal domain of Type I or the inactivated (by site-directed mutation) N-terminal half of Type II hexokinase. Virtually no activity was seen with chimeras constructed with the N-terminal half of the Type III isozyme and catalytically inactive (by site-directed mutation) C-terminal halves of Type I or Type II hexokinase. Substrate inhibition by Glc is seen only with the Type III isozyme and with chimeric forms containing the C-terminal half of Type III hexokinase and the N-terminal half of Type I or Type II isozyme, the latter inactivated by site-directed mutation; this is attributed to conformational changes induced by binding of Glc to a low affinity site in the N-terminal half, with subsequent effect on catalytic activity of the C-terminal half. These results also provide further insight into the role of interactions (or lack of interactions) between the N- and C-terminal halves in the inhibition of the Type I-III isozymes by Glc-6-P, its antagonism by low concentrations of Pi, and the inhibition seen at higher concentrations of Pi.
先前的研究表明，催化功能与哺乳动物己糖激酶II型同工酶的两个部分均相关，而I型同工酶在功能上分化为催化性的C末端部分和调节性的N末端部分。现已证明，III型同工酶在功能组织上与I型同工酶相似。由III型己糖激酶的N末端部分与I型或II型己糖激酶的C末端部分组成的嵌合体具有可归因于C末端部分的活性，并且其活性与由III型的C末端部分和I型的内在无活性的N末端结构域或II型己糖激酶的（通过定点突变）失活的N末端部分组成的嵌合体相似。用III型同工酶的N末端部分与I型或II型己糖激酶的催化无活性（通过定点突变）的C末端部分构建的嵌合体几乎没有活性。仅在III型同工酶以及含有III型己糖激酶C末端部分和I型或II型同工酶N末端部分（后者通过定点突变失活）的嵌合形式中观察到葡萄糖对底物的抑制作用；这归因于葡萄糖与N末端部分中的低亲和力位点结合诱导的构象变化，随后对C末端部分的催化活性产生影响。这些结果还进一步深入了解了N末端和C末端部分之间的相互作用（或缺乏相互作用）在葡萄糖-6-磷酸对I-III型同工酶的抑制作用、低浓度无机磷酸对其的拮抗作用以及高浓度无机磷酸时所观察到的抑制作用中的作用。
