Smith A D, Wilson J E
Department of Biochemistry, Michigan State University, East Lansing 48824.
Arch Biochem Biophys. 1991 Nov 15;291(1):59-68. doi: 10.1016/0003-9861(91)90105-r.
The Type I isozyme of rat hexokinase (ATP:D-hexose 6-phosphotransferase, EC 2.7.1.1) is comprised of N- and C-terminal domains, associated with regulatory and catalytic functions, respectively. Extensive sequence similarity between the domains is consistent with evolution of the enzyme by gene duplication and fusion. Cleavage at tryptic sites located in the C-terminal domain is markedly sensitive to ligands present during digestion, while analogous sites in the N-terminal domain are either resistant to trypsin or unaffected by the presence of ligands. These results imply a lack of structural equivalence between the N- and C-terminal domains, with the overall structure of the N-terminal domain being "tighter" and with a major component of ligand-induced conformational changes being focused in the C-terminal domain. Based on a previously proposed structure for brain hexokinase, protection by substrate hexoses is attributed to substrate-induced closing of a cleft in the C-terminal domain. Similar protection at C-terminal cleavage sites results from binding of inhibitory hexose-6-phosphates to the N-terminal domain. In addition, hexose-6-phosphates evoke cleavage at a site, T5, located in a region that has been associated with binding of ATP to the C-terminal domain. Thus, alterations in this region, coupled with reduced accessibility resulting from cleft closure, may account for the mutually exclusive binding of inhibitory hexose-6-phosphates and substrate ATP. In the absence of Mg2+, all nucleoside triphosphates examined (ATP, UTP, CTP, and GTP) protected against digestion by trypsin. In contrast, ATP-Mg2+ stabilized the C-terminal domain but destabilized the N-terminal domain, while the chelated forms of the other nucleoside triphosphates were similar to the unchelated forms in their effect on proteolysis; the unique response to ATP-Mg2+ reflects the specificity for ATP as a substrate.
大鼠己糖激酶I型同工酶（ATP：D-己糖6-磷酸转移酶，EC 2.7.1.1）由N端和C端结构域组成，分别与调节功能和催化功能相关。这些结构域之间广泛的序列相似性与该酶通过基因复制和融合的进化过程相一致。位于C端结构域的胰蛋白酶切割位点对消化过程中存在的配体非常敏感，而N端结构域中的类似位点对胰蛋白酶具有抗性或不受配体存在的影响。这些结果表明N端和C端结构域之间缺乏结构等效性，N端结构域的整体结构更“紧密”，配体诱导的构象变化的主要部分集中在C端结构域。基于先前提出的脑己糖激酶结构，底物己糖的保护作用归因于底物诱导的C端结构域裂隙的闭合。C端切割位点的类似保护作用是由抑制性己糖-6-磷酸与N端结构域的结合引起的。此外，己糖-6-磷酸在位于与ATP结合到C端结构域相关区域的T5位点引发切割。因此，该区域的改变，加上裂隙闭合导致的可及性降低，可能解释了抑制性己糖-6-磷酸和底物ATP的相互排斥结合。在没有Mg2+的情况下，所检测的所有核苷三磷酸（ATP、UTP、CTP和GTP）都能保护免受胰蛋白酶消化。相比之下，ATP-Mg2+稳定了C端结构域但使N端结构域不稳定，而其他核苷三磷酸的螯合形式在对蛋白水解的影响方面与未螯合形式相似；对ATP-Mg2+的独特反应反映了对ATP作为底物的特异性。
