Barnakov Alexander N, Barnakova Ludmila A, Hazelbauer Gerald L
Washington State University, Pullman 99164-4660, USA.
J Biol Chem. 2002 Nov 1;277(44):42151-6. doi: 10.1074/jbc.M206245200. Epub 2002 Aug 23.
Sensory adaptation in bacterial chemotaxis is mediated by covalent modification of chemoreceptors. Specific glutamyl residues are methylated and demethylated in reactions catalyzed by methyltransferase CheR and methylesterase CheB. In Escherichia coli and Salmonella enterica serovar typhimurium, efficient adaptational modification by either enzyme is dependent on a conserved pentapeptide sequence at the chemoreceptor carboxyl terminus, a position distant from the sites of modification. For CheR-catalyzed methylation, previous work demonstrated that this sequence acts as a high affinity docking site, enhancing methylation by increasing enzyme concentration near methyl-accepting glutamates. We investigated pentapeptide-mediated enhancement of CheB-catalyzed demethylation and found it occurred by a distinctly different mechanism. Assays of binding between CheB and the pentapeptide sequence showed that it was too weak to have a significant effect on local enzyme concentration. Kinetic analyses revealed that interaction of the sequence and the methylesterase enhanced the rate constant of demethylation not the Michaelis constant. This allosteric activation occurred if the sequence was attached to chemoreceptor, but hardly at all if it was present as an isolated peptide. In addition, free peptide inhibited demethylation of the native receptor carrying the pentapeptide sequence at its carboxyl terminus. These observations imply that the allosteric change is transmitted through the protein substrate, not the enzyme.
细菌趋化作用中的感官适应是由化学感受器的共价修饰介导的。特定的谷氨酰残基在甲基转移酶CheR和甲基酯酶CheB催化的反应中发生甲基化和去甲基化。在大肠杆菌和鼠伤寒沙门氏菌中，这两种酶的有效适应性修饰都依赖于化学感受器羧基末端的一个保守五肽序列，该位置远离修饰位点。对于CheR催化的甲基化，先前的研究表明，这个序列作为一个高亲和力的对接位点，通过增加甲基接受性谷氨酸附近的酶浓度来增强甲基化。我们研究了五肽介导的CheB催化的去甲基化增强作用，发现其发生机制明显不同。CheB与五肽序列之间的结合分析表明，其结合力太弱，对局部酶浓度没有显著影响。动力学分析表明，该序列与甲基酯酶的相互作用增强了去甲基化的速率常数，而不是米氏常数。如果该序列连接到化学感受器上，就会发生这种变构激活，但如果它以分离的肽形式存在，则几乎不会发生。此外，游离肽抑制了在其羧基末端携带五肽序列的天然受体的去甲基化。这些观察结果表明，变构变化是通过蛋白质底物传递的，而不是通过酶传递的。
