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

An open reading frame (ORF) of snake muscle fructose-l,6-bisphosphatase (Fr u-1,6-P(2)ase) was obtained by the RT-PCR method with degenerate primers, f ollowed by RACE-PCR. The cDNA of Fru-1,6-P(2)ase, 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 enz yme, including inhibition behavior by AMP, were identical to that of the ti ssue form. Replacement of the N-terminal sequence of this enzyme by the cor responding region of rat liver Fru1,6-P(2)ase shows that the activity was f ully retained in the chimeric enzyme. The inhibition constant (K-i) of AMP at pH 7.5, however, increases sharply from 0.85 mu M (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 activat ed at alkaline pH by AMP indicated that the AMP activation requires the glo bal structure beyond the area.