title: Molecular cloning, expression and purification of muscle fructose-1,6-bisphosphatase from Zaocys dhumnades: the role of the N-terminal sequence in AMP activation at alkaline pH.
abstract: An open reading frame (ORF) of snake muscle fructose-1,6-bisphosphatase (Fru-1,6-P2ase) was obtained by the RT-PCR method with degenerate primers, followed by RACE-PCR. The cDNA of Fru-1,6-P2ase, encoding 340 amino acids, is highly homologous to that of mammalian species, especially human muscle, with a few exceptions. Kinetic parameters of the purified recombinant enzyme, including inhibition behavior by AMP, were identical to that of the tissue form. Replacement of the N-terminal sequence of this enzyme by the corresponding region of rat liver Fru-1,6-P2ase shows that the activity was fully retained in the chimeric enzyme. The inhibition constant (Ki) of AMP at pH 7.5, however, increases sharply from 0.85 microM (wild-type) to 1.2 mM (chimeric enzyme). AMP binding is mainly located in the N-terminal region, and the allosteric inhibition was shown not to be merely determined by the backbone of this region. The fact that the chimeric enzyme could be activated at alkaline pH by AMP indicated that the AMP activation requires the global structure beyond the area.
authors.affiliation: Shanghai Institute of Biochemistry, Academia Sinica, China.
authors.forename: F W
authors.initials: FW
authors.lastname: Zhang
authors.forename: F K
authors.initials: FK
authors.lastname: Zhao
authors.forename: G J
authors.initials: GJ
authors.lastname: Xu
mesh_terms: D000249:Adenosine Monophosphate / Q000378:metabolism*; D000595:Amino Acid Sequence; D000818:Animals; D001483:Base Sequence; D003001:Cloning, Molecular; D018076:DNA, Complementary; D006597:Fructose-Bisphosphatase / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism; D006863:Hydrogen-Ion Concentration; D007700:Kinetics; D008969:Molecular Sequence Data; D009132:Muscles / Q000201:enzymology*; D016366:Open Reading Frames; D017386:Sequence Homology, Amino Acid; D012911:Snakes
chemical_list: D018076:DNA, Complementary; D000249:Adenosine Monophosphate; D006597:Fructose-Bisphosphatase