Effect of ligand-induced conformational changes on the reactivity of specific sulfhydryl residues in rat brain hexokinase.
Effect of ligand-induced conformational changes on the reactivity of specific sulfhydryl residues in rat brain hexokinase.
复制标题
配体诱导的构象变化对大鼠脑己糖激酶中特定巯基残基反应性的影响。
DOI：
10.1016/0003-9861(90)90628-c
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发表时间：
1990
影响因子：
3.9
通讯作者：
Wilson,JE
中科院分区：
文献类型：
--
作者：
Hutny,J;Wilson,JE
大鼠脑己糖激酶（ATP:d-己糖6-磷酸转移酶，EC 2.7.1.1）含有21个半胱氨酸残基。根据酶的氨基酸序列，预测它们分布在由胰蛋白酶消化产生的14个肽中。这些含有半胱氨酸残基的肽中，有10个通过与特定的巯基试剂2-溴乙酰氨基-4-硝基苯酚的反应衍生出来，已在HPLC肽图中被鉴定出来；这四种缺失的肽被预测为相对较大的疏水性特征，这些特性可能在目前的条件下阻止了它们的检测。被鉴定的10个肽所包含的序列包括酶中21个半胱氨酸残基中的12个。这些巯基与2-溴乙酰氨基-4-硝基苯酚的相对反应性已经被评估过，并且大体上与基于它们在该酶先前提出的结构中的可接近性的预测一致。测定了不同配体对已识别巯基反应性的影响；由配体诱导的构象变化引起的反应性改变的独特模式已经被观察到。随着葡萄糖6-磷酸（Glc-6-P）或pi浓度的增加，观察到双相效应。在这两种情况下，在低浓度的配体下，分子n端一半的巯基的反应性降低，而配体浓度的进一步增加导致c端一半的巯基的反应性降低。相比之下，N端和c端半段的巯基同时受到Glc浓度增加的保护。这些结果与先前的研究一致，表明(a)存在两个与Glc-6- p orPi结合的位点，一个高亲和力位点在n端一半，一个亲和力较低的位点在c端一半，以及(b) Glc与位于c端一半的单个位点结合，但在整个分子中引起构象效应；葡萄糖的类似物，n -乙酰氨基葡萄糖，先前被证明对构象的影响更有限，只影响分子c端一半的巯基的反应性。正如对巯基反应性的影响所反映的那样，核苷酸结合引起的构象变化明显取决于嘌呤或嘧啶碱基的特定性质以及多磷酸侧链的长度和螯合状态。这些结果将注意力集中在受这些配体结合影响的分子的特定区域（巯基的直接环境）上。
Rat brain hexokinase (ATP:d-hexose 6-phosphotransferase, EC 2.7.1.1) contains 21 cysteine residues. On the basis of the amino acid sequence of the enzyme, these are predicted to be distributed among 14 peptides produced by tryptic digestion. Ten of these peptides, containing cysteine residues derivatized by reaction with the specific sulfhydryl reagent 2-bromoacetamido-4-nitrophenol have been identified in HPLC peptide maps; the four missing peptides are predicted to be relatively large and hydrophobic in character, properties that may have prevented their detection under the present conditions. The sequences encompassed by the 10 identified peptides include 12 of the 21 cysteine residues in the enzyme. The relative reactivity of these sulfhydryl groups with 2-bromoacetamido-4-nitrophenol has been assessed, and is in general accord with what might be predicted on the basis of their accessibility in the previously proposed structure for this enzyme. The effect of various ligands on reactivity of identified sulfhydryl groups has been determined; unique patterns of altered reactivity, resulting from ligand-induced conformational changes, have been observed. Biphasic effects were observed with increasing concentrations of either glucose 6-phosphate (Glc-6-P) orPi. In both cases, decreased reactivity of sulfhydryls in the N-terminal half of the molecule was observed at low concentrations of the ligand, while further increase in ligand concentration resulted in decreased reactivity of sulfhydryl groups in the C-terminal half. In contrast, sulfhydryls in both N- and C-terminal halves were protected concomitantly by increasing concentrations of Glc. These results are consistent with previous studies that indicated (a) the existence of two sites for binding of Glc-6-P orPi, a high affinity site in the N-terminal half and a site with lower affinity in the C-terminal half of the brain hexokinase molecule, and (b) binding of Glc to a single site located in the C-terminal half but evoking conformational effects throughout the molecule; the glucose analog,N-acetylglucosamine, previously shown to have more limited effects on conformation, affected reactivity of sulfhydryl groups only in the C-terminal half of the molecule. As reflected by effects on reactivity of sulfhydryl groups, conformational changes induced by binding of nucleotides depends markedly on the specific nature of the purine or pyrimidine base as well as the length and chelation status of the polyphosphate side chain. These results focus attention on specific regions of the molecule (the immediate environment of the sulfhydryl groups) that are affected by the binding of these ligands.