The HPr kinase from Bacillus subtilis is a homo-oligomeric enzyme which exhibits strong positive cooperativity for nucleotide and fructose 1,6-bisphosphate binding
The HPr kinase from Bacillus subtilis is a homo-oligomeric enzyme which exhibits strong positive cooperativity for nucleotide and fructose 1,6-bisphosphate binding
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DOI：
10.1074/jbc.275.3.1773
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发表时间：
2000-01-21
影响因子：
4.8
通讯作者：
Galinier, A
中科院分区：
文献类型：
--
作者：
Jault, JM;Fieulaine, S;Galinier, A
碳分解代谢物阻遏允许细菌响应可代谢碳源的可用性而快速改变分解代谢基因的表达。在枯草芽孢杆菌中，这种现象由Hh激酶（HprK）控制，Hh激酶催化残基Ser-46上HPr（含组氨酸的蛋白质）或Crh（分解代谢物阻遏HPr）的ATP依赖性磷酸化。本文报道了B，subtilis HprK形成的同源寡聚体最有可能由八个亚基组成。与这种复杂的结构相关，该酶显示出与其变构激活剂果糖1，6-二磷酸结合的强正协同性，这可以通过其磷酸化活性的动力学或其独特的色氨酸残基Trp-235的固有荧光特性来证明，进一步表明，1，6-二磷酸果糖对HPr磷酸化的激活基本上发生在低ATP和酶浓度下。在磷酸化或荧光实验中，天然核苷酸或其2 '（3'）-N-甲基邻氨基苯甲酰衍生物的结合也检测到正协同性。最有趣的是，通过使用碘化物或丙烯酰胺淬灭HprK色氨酸荧光揭示了低聚物群体内色氨酸残基的异质性，这表明该酶存在于两种不同的构象中。这一结果表明，HprK所显示的正协同催化机制的协调对称模型。
Carbon catabolite repression allows bacteria to rapidly alter the expression of catabolic genes in response to the availability of metabolizable carbon sources. In Bacillus subtilis, this phenomenon is controlled by the Hh kinase (HprK) that catalyzes ATP-dependent phosphorylation of either HPr (histidine containing protein) or Crh (catabolite repression HPr) on residue Ser-46. We report here that B, subtilis HprK forms homo-oligomers constituted most likely of eight subunits, Related to this complex structure, the enzyme displays strong positive cooperativity for the binding of its allosteric activator, fructose 1,6-bisphosphate, as evidenced by either kinetics of its phosphorylation activity or the intrinsic fluorescence properties of its unique tryptophan residue, Trp-235, It is further shown that activation of HPr phosphorylation by fructose 1,6-bisphosphate essentially occurs at low ATP and enzyme concentrations. A positive cooperativity was also detected for the binding of natural nucleotides or their 2'(3')-N-methylanthraniloyl derivatives, in either phosphorylation or fluorescence experiments. Most interestingly, quenching of the HprK tryptophan fluorescence by using either iodide or acrylamide revealed a heterogeneity of tryptophan residues within the population of oligomers, suggesting that the enzyme exists in two different conformations. This result suggests a concerted-symmetry model for the catalytic mechanism of positive cooperativity displayed by HprK.