Mutations in the hinge of a dynamic loop broadly influence functional properties of fructose-1,6-bisphosphatase

Loop 52-72 of porcine fructose-1,6-bisphosphatase may play a central role i n the mechanism of catalysis and allosteric inhibition by AMP. The loop piv ots between different conformational states about a hinge located at residu es 50 and 51. The insertion of proline separately at positions 50 and 51 re duces k(cat) by up to 3-fold, with no effect on the K-m for fructose 1,6-bi sphosphate. The K-a for Mg2+ in the Lys(50) --> Pro mutant increases simila r to 15-fold, whereas that for the Ala(51) --> Pro mutant is unchanged. Alt hough these mutants retain wildtype binding affinity for AMP and the fluore scent AMP analog 2'(3')-O-(trinitrophenyl) adenosine 5'-monophosphate, the K-i for AMP increases 8000- and 280-fold in the position 50 and 51 mutants, respectively. In fact, the mutation Lys(50) --> Pro changes the mechanism of AMP inhibition with respect to Mg2+ from competitive to noncompetitive a nd abolishes K+ activation. The K-i for fructose 2,6-bisphosphate increases similar to 20- and 30-fold in the Lys(50) --> Pro and Ala(51) --> Pro muta nts, respectively. Fluorescence from a tryptophan introduced by the mutatio n of Tyr(57) suggests an altered conformational state for Loop 52-72 due to the proline at position 50. Evidently, the Pro(50) mutant binds AMP with h igh affinity at the allosteric site, but the mechanism of allosteric regula tion of catalysis has been disabled.