THE ALLOSTERIC SITE OF HUMAN LIVER FRUCTOSE-1,6-BISPHOSPHATASE - ANALYSIS OF 6 AMP SITE MUTANTS BASED ON THE CRYSTAL-STRUCTURE

The molecular structure of human liver fructose-1,6-bisphosphatase com plexed with AMP was determined by x-ray diffraction using molecular re placement, starting from the pig kidney enzyme AMP complex. Of the 34 amino acid residues which differ between these two sequences, only one interacts with AMP; Met(30) in pig kidney is Leu(30) in human liver. From this analysis, six sites in which side chains of amino acid resid ues are in contact with AMP Ala(24), Leu(30), Thr(31), Tyr(113), Arg(1 40), and Met(177), were mutated by polymerase chain reaction. The wild -type and mutant forms were expressed in Escherichia coli, purified, a nd their kinetic properties determined. Circular dichroism spectra of the mutants were indistinguishable from that of the wild-type enzyme. Kinetic analyses revealed that all forms had similar turnover numbers, K-m values for fructose 2,6-bisphosphate, and inhibition constants fo r fructose 2,6-bisphosphate. Apparent K-i values for AMP inhibition of the Leu(30) --> Phe and Met(177) --> Ala mutants were similar to thos e of the wild-type enzyme, but the apparent K-i values for the Arg(140 ) --> Ala and Ala(24) --> Phe mutants were 7-to 20-fold higher, respec tively. The Thr(31) --> Ser mutant exhibited a 5-fold increase in appa rent K-i for AMP, while mutation of Thr(31) to Ala increased the appar ent K-i 120-fold. AMP inhibition of the Tyr(113) --> Phe mutant was un detectable even at millimolar AMP concentrations. Fructose 2,6-bisphos phate potentiated AMP inhibition of the mutants to the same extent as for the wild-type enzyme, except in the case of the Thr(31) --> Ala an d Tyr(113) --> Phe mutants. Thus, the Met(177) --> Ala mutant suggests that the side chain beyond C alpha is not needed for AMP binding, and that the Leu(30) --> Phe mutant preserves the AMP contacts with these side chains. Thr(31), Tyr(113), and Arg(140) form key hydrogen bonds to AMP consistent with strong side chain interactions in the wild-type enzyme. Finally, the absence of any effect of fructose 2,6-bisphospha te on AMP inhibition observed in the Thr(31) --> Ala mutant may be an important clue relating to the mechanism of synergism of these two inh ibitors.