Yi X, Weis R M
Department of Chemistry, University of Massachusetts, Box 34510, Amherst, MA 01003-9336, USA.
Biochim Biophys Acta. 2002 Apr 1;1596(1):28-35. doi: 10.1016/s0167-4838(01)00314-4.
To mediate adaptation to stimuli, the methyltransferase (CheR) catalyzes methyl group transfer from S-adenosyl-L-methionine (SAM) to glutamyl residues in the transmembrane receptors of the bacterial chemosensory signaling pathway. The interaction between receptors and CheR occurs at two sites: a methylation site-active site interaction, and a 'docking' site interaction that is separated both from the methylation sites and the CheR active site. It is not certain if the docking site interaction functions merely to localize the transferase in close proximity to the methylation sites, or if it also increases CheR catalytic activity. Isothermal titration calorimetry experiments are conducted to test for allosteric interactions between the docking and active sites on CheR, which are expected to be present if docking activates CheR. The binding parameters (DeltaG, DeltaH, DeltaS) of a substrate analog of SAM, S-adenosyl-L-homocysteine (SAH), are measured both in the absence and presence of saturating concentrations of a pentapeptide (NWETF) that defines the docking receptor docking segment. SAH binding is unaffected by the presence of saturating NWETF, providing evidence that an allosteric activation of CheR does not take place upon docking, and thus supports the idea that the CheR-NWETF interaction merely functions to localize CheR near the sites of methylation.
为介导对刺激的适应性，甲基转移酶（CheR）催化甲基从S-腺苷-L-甲硫氨酸（SAM）转移至细菌化学感应信号通路跨膜受体中的谷氨酰残基。受体与CheR之间的相互作用发生在两个位点：一个甲基化位点-活性位点相互作用，以及一个与甲基化位点和CheR活性位点均分离的“对接”位点相互作用。尚不确定对接位点相互作用仅仅是为了将转移酶定位在靠近甲基化位点的位置，还是它也会增加CheR的催化活性。进行等温滴定量热法实验以测试CheR上对接位点与活性位点之间的变构相互作用，如果对接激活CheR，预计会存在这种相互作用。在不存在和存在定义对接受体对接片段的五肽（NWETF）饱和浓度的情况下，均测量SAM的底物类似物S-腺苷-L-高半胱氨酸（SAH）的结合参数（ΔG、ΔH、ΔS）。SAH的结合不受饱和NWETF存在的影响，这表明对接时CheR不会发生变构激活，因此支持了CheR-NWETF相互作用仅仅起到将CheR定位在甲基化位点附近的作用这一观点。
