EFFECTOR-MEDIATED STIMULATION OF ATPASE ACTIVITY BY THE SIGMA(54)-DEPENDENT TRANSCRIPTIONAL ACTIVATOR FHLA FROM ESCHERICHIA-COLI
EFFECTOR-MEDIATED STIMULATION OF ATPASE ACTIVITY BY THE SIGMA(54)-DEPENDENT TRANSCRIPTIONAL ACTIVATOR FHLA FROM ESCHERICHIA-COLI
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DOI：
10.1128/jb.177.10.2798-2803.1995
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发表时间：
1995-05-01
影响因子：
3.2
通讯作者：
BOCK, A
中科院分区：
文献类型：
--
作者：
HOPPER, S;BOCK, A
FHLA蛋白是来自大肠杆菌的甲酸调节子基因的转录调节因子，该蛋白与NTRC家族的σ（54）依赖性调节因子在中心和C-末端结构域中具有同源性，但不同之处在于具有缺少作为磷酸化位点的天冬氨酸残基的延伸的N末端。纯化的FHLA显示出固有的ATP酶活性，其被甲酸盐和DNA微弱地刺激。甲酸盐和携带FHLA结合的上游调节序列的DNA的存在导致ATP水解速率的大幅增加，次磷酸盐（甲酸盐的结构类似物）和叠氮化物（甲酸盐的过渡态类似物）也刺激ATP酶活性，支持甲酸盐是FHLA的直接配体的结论，FHLA的半最大饱和，甲酸盐发生在约5 mM，靶DNA的半最大饱和发生在约50 nM。甲酸盐对ATP酶活性的刺激是通过ATP的表观Km的降低来实现的，而DNA结合位点的作用也影响反应的K-cat。与FHLA的ATP切割竞争，表明至少它们与FHLA结合。FHLA的特异性ATP酶活性依赖于测定中FHLA的浓度，特别是在DNA和甲酸盐存在下。因此，效应物的直接连接导致与NTRC型调节剂的磷酸化相同的结果，即刺激ATP酶活性。
The FHLA protein is the transcriptional regulator of the genes of the formate regulon from Escherichia coli, The protein shares homology with the sigma(54)-dependent regulators of the NTRC family in the central and C-terminal domains but differs in possessing an extended N terminus lacking the aspartate residue which is the site of phosphorylation. Purified FHLA displays intrinsic ATPase activity which is stimulated weakly by formate and DNA, The presence of both formate and DNA carrying the upstream regulatory sequence to which FHLA binds leads to a large increase in the rate of ATP hydrolysis, Hypophosphite, a structural analog of formate, and azide, a transition state analog of formate, also stimulate ATPase activity, supporting the conclusion that formate is a direct ligand of FHLA, Half-maximal saturation of FHLA,vith formate took place at around 5 mM, and half-maximal saturation with target DNA took place at around 50 nM, The stimulation of ATPase activity by formate was conferred by a decrease in the apparent K-m for ATP, whereas the effect of the DNA binding site also affected the K-cat of the reaction, The other nucleoside triphosphates, GTP, UTP, and CTP, competed with ATP cleavage by FHLA, suggesting at least their binding to FHLA. The specific ATPase activity of FHLA was dependent on the concentration of FHLA in the assay, especially in the presence of DNA and formate, Direct liganding of the effector, therefore, leads to the same consequence as phosphorylation for the NTRC-type regulators, namely, stimulation of ATPase activity.