Rat brain hexokinase: location of the substrate hexose binding site in a structural domain at the C-terminus of the enzyme.
Rat brain hexokinase: location of the substrate hexose binding site in a structural domain at the C-terminus of the enzyme.
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大鼠脑己糖激酶：酶 C 末端结构域中底物己糖结合位点的位置。
DOI：
10.1016/0003-9861(87)90116-0
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发表时间：
1987
影响因子：
3.9
通讯作者：
Wilson,JE
中科院分区：
文献类型：
--
作者：
Schirch,DM;Wilson,JE
葡萄糖类似物 N-（溴乙酰基）-d-葡萄糖胺 (GlcNBrAc) 以前用于标记 II 型大鼠肌肉和 I 型牛脑己糖激酶的葡萄糖结合位点，也可通过伪一级动力学使大鼠脑己糖激酶（ATP：d-己糖 6-磷酸转移酶，EC 2.7.1.1）失活。失活主要通过涉及己糖激酶和 GlcNBrAc 之间形成复合物的“特异性”途径发生，但也会发生显着的非特异性（即，没有事先形成复合物）失活，并且导出了描述这种行为的方程。失活取决于具有碱性 pKa 的残基的去质子化，与报道的牛脑己糖激酶的情况一致，修饰残基是巯基。亲和标记以无法区分的速率修饰三个残基（每个酶分子），但这些残基中只有一个似乎对活性至关重要。修饰酶的氨基酸分析表明三个半胱氨酸残基衍生化；没有迹象表明其他残基可能与卤代乙酰衍生物发生反应。动力学分析和保护性配体的作用与葡萄糖结合位点上关键的巯基的位置一致。肽图谱技术允许在酶 C 末端的 40 kDa 结构域中定位关键残基，从而定位葡萄糖结合位点。这与最近显示的包含 ATP 结合位点的域相同。因此，催化功能被分配给大鼠脑己糖激酶的C末端结构域。
A glucose analog,N-(bromoacetyl)-d-glucosamine (GlcNBrAc), previously used to label the glucose binding sites of rat muscle Type II and bovine brain Type I hexokinases, also inactivates rat brain hexokinase (ATP:d-hexose 6-phosphotransferase, EC 2.7.1.1) with pseudo-first-order kinetics. Inactivation occurs predominantly via a “specific” pathway involving formation of a complex between hexokinase and GlcNBrAc, but significant nonspecific (i.e., without prior complex formation) inactivation also occurs, and equations to describe this behavior are derived. Inactivation is dependent on deprotonation of a residue with an alkaline pKa, consistent with the modified residue being a sulfhydryl group as reported to be the case with the hexokinase of bovine brain. The affinity label modifies three residues (per molecule of enzyme) at indistinguishable rates, but only one of these residues appears to be critical for activity. Amino acid analysis of the modified enzyme indicates derivatization of three cysteine residues; there was no indication of modification of other residues potentially reactive with haloacetyl derivatives. Kinetic analysis and effects of protective ligands were consistent with location of the critical sulfhydryl at the glucose binding site. Peptide mapping techniques permitted localization of the critical residue, and thus the glucose binding site, in a 40-kDa domain at the C-terminus of the enzyme. This is the same domain recently shown to include the ATP binding site. Thus, catalytic function is assigned to the C-terminal domain of rat brain hexokinase.
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