Hopper S, Böck A
Lehrstuhl für Mikrobiologie, Universität München, Germany.
J Bacteriol. 1995 May;177(10):2798-803. doi: 10.1128/jb.177.10.2798-2803.1995.
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 with 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 Km for ATP, whereas the effect of the DNA binding site also affected the Kcat 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.
FHLA蛋白是大肠杆菌甲酸调节子基因的转录调节因子。该蛋白在中央结构域和C端结构域与NTRC家族的σ54依赖性调节因子具有同源性，但不同之处在于其N端延伸，缺少作为磷酸化位点的天冬氨酸残基。纯化的FHLA表现出内在的ATP酶活性，该活性受到甲酸和DNA的微弱刺激。同时存在甲酸和携带FHLA结合的上游调节序列的DNA会导致ATP水解速率大幅增加。次磷酸盐（甲酸的结构类似物）和叠氮化物（甲酸的过渡态类似物）也刺激ATP酶活性，支持了甲酸是FHLA直接配体的结论。FHLA与甲酸的半数最大饱和度发生在约5 mM，与靶DNA的半数最大饱和度发生在约50 nM。甲酸对ATP酶活性的刺激是由于ATP的表观Km降低所致，而DNA结合位点的作用也影响了反应的催化常数（Kcat）。其他核苷三磷酸，即GTP、UTP和CTP，与FHLA催化的ATP裂解竞争，表明它们至少与FHLA结合。FHLA的特异性ATP酶活性取决于测定中FHLA的浓度，特别是在存在DNA和甲酸的情况下。因此，效应物的直接配体作用与NTRC型调节因子的磷酸化导致相同的结果，即刺激ATP酶活性。
