MAJOR CHANGES IN THE KINETIC MECHANISM OF AMP INHIBITION AND AMP COOPERATIVITY ATTEND THE MUTATION OF ARG(49) IN FRUCTOSE-1,6-BISPHOSPHATASE

The significance of subunit interface residues Arg(49) and Lys(50) in the function of porcine liver fructose-1,6-bisphosphatase was explored by site-directed mutagenesis, initial rate kinetics, and circular dic hroism spectroscopy. The Lys(50) --> Met mutant had kinetic properties similar to the wild-type enzyme but was more thermostable. Mutants Ar g(49) --> Leu, Arg(49) --> Asp, Arg(49) --> Cys were less thermostable than the wild-type enzyme yet exhibited wild-type values for k(cat) a nd K-m. The K-i for the competitive inhibitor fructose 2, 6-bisphospha te increased 3- and 8-fold in Arg(49) --> Leu and Arg(49) --> Asp, res pectively, The K-a for Mg2+ increased 4-8-fold for the Arg(49) mutants , with no alteration in the cooperativity of Mg2+ binding, Position 49 mutants had 4-10-fold lower AMP affinity, Most significantly, the mec hanism of AMP inhibition with respect to fructose 1,6-bisphosphate cha nged from noncompetitive (wild-type enzyme) to competitive (Arg(49) -- > Leu and Arg(49) --> ASP mutants) and to uncompetitive (Arg(49) --> C ys mutant), In addition, AMP cooperativity was absent in the Arg(49) m utants, The R and T-state circular dichroism spectra of the position 4 9 mutants were identical and superimposable on only the R-state spectr um of the wild-type enzyme. Changes from noncompetitive to competitive inhibition by AMP can be accommodated within the framework of a stead y-state Random Ri Ri mechanism, The appearance of uncompetitive inhibi tion, however, suggests that a more complex mechanism may be necessary to account for the kinetic properties of the enzyme.