title: New insight into the binding modes of TNP-AMP to human liver fructose-1,6-bisphosphatase.
abstract: Human liver fructose-1,6-bisphosphatase (FBPase) contains two binding sites, a substrate fructose-1,6-bisphosphate (FBP) active site and an adenosine monophosphate (AMP) allosteric site. The FBP active site works by stabilizing the FBPase, and the allosteric site impairs the activity of FBPase through its binding of a nonsubstrate molecule. The fluorescent AMP analogue, 2',3'-O-(2,4,6-trinitrophenyl)adenosine 5'-monophosphate (TNP-AMP) has been used as a fluorescent probe as it is able to competitively inhibit AMP binding to the AMP allosteric site and, therefore, could be used for exploring the binding modes of inhibitors targeted on the allosteric site. In this study, we have re-examined the binding modes of TNP-AMP to FBPase. However, our present enzyme kinetic assays show that AMP and FBP both can reduce the fluorescence from the bound TNP-AMP through competition for FBPase, suggesting that TNP-AMP binds not only to the AMP allosteric site but also to the FBP active site. Mutagenesis assays of K274L (located in the FBP active site) show that the residue K274 is very important for TNP-AMP to bind to the active site of FBPase. The results further prove that TNP-AMP is able to bind individually to the both sites. Our present study provides a new insight into the binding mechanism of TNP-AMP to the FBPase. The TNP-AMP fluorescent probe can be used to exam the binding site of an inhibitor (the active site or the allosteric site) using FBPase saturated by AMP and FBP, respectively, or the K247L mutant FBPase.
authors.affiliation: Key Laboratory of Pesticide & Chemical Biology (CCNU), Ministry of Education, College of Chemistry, Central China Normal University, Wuhan 430079, China.
authors.forename: Xinya
authors.initials: X
authors.lastname: Han
authors.affiliation: Key Laboratory of Pesticide & Chemical Biology (CCNU), Ministry of Education, College of Chemistry, Central China Normal University, Wuhan 430079, China.
authors.forename: Yunyuan
authors.initials: Y
authors.lastname: Huang
authors.affiliation: College of Chemistry and Molecular Sciences, Wuhan University, Wuhan 430072, China.
authors.forename: Rui
authors.initials: R
authors.lastname: Zhang
authors.affiliation: Key Laboratory of Pesticide & Chemical Biology (CCNU), Ministry of Education, College of Chemistry, Central China Normal University, Wuhan 430079, China.
authors.forename: San
authors.initials: S
authors.lastname: Xiao
authors.affiliation: Key Laboratory of Pesticide & Chemical Biology (CCNU), Ministry of Education, College of Chemistry, Central China Normal University, Wuhan 430079, China.
authors.forename: Shuaihuan
authors.initials: S
authors.lastname: Zhu
authors.affiliation: Key Laboratory of Pesticide & Chemical Biology (CCNU), Ministry of Education, College of Chemistry, Central China Normal University, Wuhan 430079, China.
authors.forename: Nian
authors.initials: N
authors.lastname: Qin
authors.affiliation: Key Laboratory of Pesticide & Chemical Biology (CCNU), Ministry of Education, College of Chemistry, Central China Normal University, Wuhan 430079, China.
authors.forename: Zongqin
authors.initials: Z
authors.lastname: Hong
authors.affiliation: Key Laboratory of Pesticide & Chemical Biology (CCNU), Ministry of Education, College of Chemistry, Central China Normal University, Wuhan 430079, China.
authors.forename: Lin
authors.initials: L
authors.lastname: Wei
authors.affiliation: Key Laboratory of Pesticide & Chemical Biology (CCNU), Ministry of Education, College of Chemistry, Central China Normal University, Wuhan 430079, China.
authors.forename: Jiangtao
authors.initials: J
authors.lastname: Feng
authors.affiliation: Key Laboratory of Pesticide & Chemical Biology (CCNU), Ministry of Education, College of Chemistry, Central China Normal University, Wuhan 430079, China.
authors.forename: Yanliang
authors.initials: Y
authors.lastname: Ren
authors.affiliation: Key Laboratory of Pesticide & Chemical Biology (CCNU), Ministry of Education, College of Chemistry, Central China Normal University, Wuhan 430079, China. Electronic address: fll708@mail.ccnu.edu.cn.
authors.forename: Lingling
authors.initials: L
authors.lastname: Feng
authors.affiliation: Key Laboratory of Pesticide & Chemical Biology (CCNU), Ministry of Education, College of Chemistry, Central China Normal University, Wuhan 430079, China. Electronic address: jianwan@mail.ccnu.edu.cn.
authors.forename: Jian
authors.initials: J
authors.lastname: Wan
mesh_terms: D000249:Adenosine Monophosphate / Q000031:analogs & derivatives* / Q000378:metabolism; D000495:Allosteric Site; D020134:Catalytic Domain; D004353:Drug Evaluation, Preclinical; D005456:Fluorescent Dyes / Q000378:metabolism*; D006597:Fructose-Bisphosphatase / Q000037:antagonists & inhibitors / Q000378:metabolism*; D006801:Humans; D062105:Molecular Docking Simulation; D011485:Protein Binding; D013050:Spectrometry, Fluorescence
chemical_list: D005456:Fluorescent Dyes; D000249:Adenosine Monophosphate; C036744:2',3'-O (2,4,6-trinitrocyclohexadienylidine)adenosine 5'-monophosphate; D006597:Fructose-Bisphosphatase
keywords: Active site; Allosteric site; Fructose-1,6-bisphosphatase; Inhibitor binding site; TNP-AMP binding site