Studies on the binding of nucleotides by rat brain hexokinase.
Studies on the binding of nucleotides by rat brain hexokinase.
复制标题
大鼠脑己糖激酶结合核苷酸的研究。
DOI：
10.1016/0003-9861(82)90375-7
复制
发表时间：
1982
影响因子：
3.9
通讯作者：
Wilson,JE
中科院分区：
文献类型：
--
作者：
Baijal,M;Wilson,JE
比较了各种二磷酸和三磷酸核苷保护大鼠脑己糖激酶（ATP：d-己糖 6-磷酸转移酶，EC 2.7.1.1）活性免受胰凝乳蛋白酶、戊二醛、热和 5,5'-二硫代双（2-硝基苯甲酸）酸灭活的能力。在这些比较中，ATP 可以与其他三磷酸核苷区分开来，这可能与 ATP 作为底物的特异性有关。所有检查的核苷衍生物都提供了针对两种或多种上述灭活剂的实质性保护，表明脑己糖激酶对核苷酸的结合相对非特异性，这与核苷衍生物抑制该酶的类似缺乏特异性一致。 2-对甲苯胺基萘-6-磺酸盐 (TNS) 和四碘荧光素 (TIF) 的荧光通过与脑己糖激酶结合而增强。 TNS 结合不受各种相关代谢物（Glc、葡萄糖 6-磷酸、ATP）存在的影响，TNS 也不会抑制该酶。相比之下，由于添加各种核苷衍生物，结合的 TIF 的荧光显着降低（约 70%），并且 TIF 充当脑己糖激酶的竞争性抑制剂。这些观察结果与 TIF 与酶的核苷酸结合位点结合的观点一致。核苷酸不能完全取代 TIF 表明存在与催化位点分离的额外 TIF 结合位点（或多个位点），并且可能与 TNS 结合的位点相同，对催化活性没有影响。 ATP 和 ADP 饱和水平的影响不是相加的，表明两种化合物从同一位点（即共同的核苷酸结合位点）取代 TIF。 Glc、甘露糖和2-脱氧葡萄糖极大地增强了核苷酸置换TIF的能力，而果糖、半乳糖和N-乙酰氨基葡萄糖则没有，这表明己糖和核苷酸结合位点之间存在相互作用；己糖本身并不能有效取代 TIF。在前三种己糖而非后三种己糖存在的情况下，核苷酸的结合增强可以与这些己糖诱导酶中特定构象变化的相对能力直接相关。己糖本身并不能有效取代 TIF。葡萄糖 6-磷酸和 1,5-脱水葡萄糖醇 6-磷酸也可以取代 TIF，并且与核苷酸一样，观察到荧光最多减少约 70%，并且葡萄糖 6-磷酸的有效性在 Glc 存在下增强。测试的其他 6-磷酸己糖不能有效取代 TIF。 6-磷酸己糖取代 TIF 的特异性可能与其诱导酶中特定构象变化的能力相关。对结果进行了讨论，因为它们与已为该酶提出的核苷酸的动力学机制和变构调节有关。
Various nucleoside di- and triphosphates have been compared with respect to their ability to protect rat brain hexokinase (ATP:d-hexose 6-phosphotransferase, EC 2.7.1.1) activity against inactivation by chymotrypsin, glutaraldehyde, heat, and 5,5′-dithiobis(2-nitrobenzoic) acid. ATP could be distinguished from other nucleoside triphosphates in these comparisons, which may be related to the specificity with which ATP is utilized as a substrate. All nucleoside derivatives examined provided substantial protection against two or more of the above inactivating agents, indicating relatively nonspecific binding of nucleotides by brain hexokinase, consistent with a similar lack of specificity in the inhibition of this enzyme by nucleoside derivatives. The fluorescence of 2-p-toluidinylnaphthalene-6-sulfonate (TNS) and of tetraiodofluorescein (TIF) was enhanced by binding to brain hexokinase. TNS binding was not affected by the presence of various relevant metabolites (Glc, glucose 6-phosphate, ATP), nor did TNS inhibit the enzyme. In contrast, substantial (approximately 70%) decreases in the fluorescence of bound TIF resulted from the addition of various nucleoside derivatives, and TIF served as a competitive inhibitor of brain hexokinase. These observations are consistent with the view that TIF binds to a nucleotide binding site of the enzyme. The inability of nucleotides to totally displace TIF was taken to indicate the existence of an additional TIF binding site (or sites) discrete from the catalytic site, and probably identical to the site(s) at which TNS binds with no effect on catalytic activity. The effects of saturating levels of ATP and ADP werenotadditive indicating that both compounds were displacing TIF from the same site i.e., a common nucleotide binding site. Glc, mannose, and 2-deoxyglucose greatly enhanced the ability of nucleotides to displace TIF, while fructose, galactose, andN-acetylglucosamine did not, indicating the existence of interactions between hexose and nucleotide binding sites; the hexoses themselves were not effective at displacing TIF. The enhanced binding of nucleotides in the presence of the first three hexoses but not the latter three can be directly correlated with the relative ability of these hexoses to induce specific conformational changes in the enzyme. The hexoses themselves were not effective at displacing TIF. Glucose 6-phosphate and 1,5-anhydroglucitol 6-phosphate could also displace TIF, and as with the nucleotides, a maximum of approximately 70% decrease in fluorescence was observed and the effectiveness of glucose 6-phosphate was enhanced in the presence of Glc. Other hexose 6-phosphates tested were not effective at displacing TIF. The specificity with which hexose 6-phosphates displaced TIF could be correlated with their ability to induce specific conformational change in the enzyme. The results are discussed as they relate to the kinetic mechanism and allosteric regulation by nucleotides that have been proposed for this enzyme.