Rat brain hexokinase: location of the allosteric regulatory site in a structural domain at the N-terminus of the enzyme.
Rat brain hexokinase: location of the allosteric regulatory site in a structural domain at the N-terminus of the enzyme.
复制标题
大鼠脑己糖激酶：变构调节位点位于酶 N 末端结构域中。
DOI：
10.1016/0003-9861(87)90506-6
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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：
White,TK;Wilson,JE
大鼠脑己糖激酶在0.6M盐酸胍中变性后，对胰蛋白酶的水解反应非常敏感。葡萄糖6-磷酸（Glc-6-P）及其类似物1，5-脱水葡萄糖醇6-磷酸，选择性地保护分子的N-末端一半免受蛋白水解。这些化合物不保护分子的C-末端一半，也不保护酶活性; Glc类似物N-乙酰葡糖胺确实保护C-末端结构域和催化活性，但不阻止分子的N-末端一半的蛋白水解。这些结果与前人的工作[M. Nemat-Gorgani和J.E. Wilson（1986）Arch.Biochem.Bioprotein.251，97-103; D. M. Schirch和J. E. Wilson（1987）Arch.Biochem.Biopolymer.254，385-396]，证明了己糖和核苷酸底物的结合位点以及催化功能与位于酶C-末端的40-kDa结构域相关。他们进一步证明了变构效应物Glc-6-P的结合位点位于分子的N-末端一半，并且与催化位点不同。使用针对蛋白水解的保护作为结合的反映，表明N-末端区域中的Glc-6-P结合位点具有针对该酶上的变构效应位点描述的所有特征，这些特征是关于Glc-6-P的亲和力、特异性以及与分子的C-末端区域中的己糖结合位点的协同相互作用。这种催化和调节功能在分子的离散半部分中的配置与几个研究者的建议一致，即哺乳动物己糖激酶是通过编码类似于现今酵母酶的己糖激酶的祖先基因的复制和融合过程进化而来的，哺乳动物己糖激酶的调节位点是从最初的催化位点进化而来的。
After denaturation in 0.6mguanidine hydrochloride, rat brain hexokinase becomes highly susceptible to proteolysis by trypsin. Glucose 6-phosphate (Glc-6-P) and its analog, 1,5-anhydroglucitol 6-phosphate, selectively protect the N-terminal half of the molecule from proteolysis. These compounds do not protect the C-terminal half of the molecule, nor do they protect enzyme activity; the Glc analog,N-acetylglucosamine, does protect the C-terminal domain and catalytic activity, but does not prevent proteolysis of the N-terminal half of the molecule. These results are consistent with previous work [M. Nemat-Gorgani and J. E. Wilson (1986)Arch. Biochem. Biophys.251, 97–103; D. M. Schirch and J. E. Wilson (1987)Arch. Biochem. Biophys.254, 385–396] demonstrating that binding sites for both hexose and nucleotide substrates, and thus catalytic function, are associated with a 40-kDa domain located at the C-terminus of the enzyme. They further demonstrate that the binding site for the allosteric effector, Glc-6-P, lies in the N-terminal half of the molecule and is distinct from the catalytic site. Using protection against proteolysis as a reflection of binding, it is shown that the Glc-6-P binding site in the N-terminal region has all the characteristics described for the allosteric effector site on this enzyme in terms of affinity for Glc-6-P, specificity, and synergistic interactions with the hexose binding site in the C-terminal region of the molecule. This disposition of catalytic and regulatory functions in discrete halves of the molecule is consistent with suggestions by several investigators that mammalian hexokinases evolved by a process of duplication and fusion of an ancestral gene coding for a hexokinase similar to the present-day yeast enzyme, with the regulatory site of mammalian hexokinases having evolved from what was originally a catalytic site.