FRUCTOSE-1,6-BISPHOSPHATASE - ARGININE-22 IS INVOLVED IN STABILIZATION OF THE T-ALLOSTERIC STATE

A comparison of the X-ray crystallographic structures of the R and T a llosteric states [Ke, H. M., Liang, J.-Y., Zhang, Y., & Lipscomb, W. N . (1991) Biochemistry 30, 4412-4420] of the pig kidney fructose-1,6-bi sphosphatase (EC 3.1.3.11) reveals major changes in the quaternary str ucture of the enzyme upon the binding of the allosteric inhibitor AMP. This change in quaternary structure involves the breaking of one set of interactions that stabilize the R state and the formation of anothe r set of interactions that stabilize the T state of the enzyme. In par ticular, the interactions of Arg-22 with nearby amino acid residues ar e quite different in the R and T states of the enzyme. Although the cr ystallographic data suggest that intersubunit interactions such as tho se involving Arg-22 are important for stabilization of the R and/or T states, the X-ray structures do not provide direct evidence concerning the functional role of specific amino acid residues. Therefore, site- specific mutagenesis has been used to probe the function of Arg-22 in pig kidney fructose-1,6-bisphosphatase. The replacement of Arg-22 by A la results in a mutant enzyme with enhanced catalytic efficiency compa red to the wild-type, as indicated by a kinetic analysis showing a sli ghly lower K-m and increased V-max compared to the wild-type enzyme. I n addition, the substitution enhances both substrate inhibition and th e affinity of the inhibitor fructose 2,6-bisphosphate. Moreover, the r eplacement of Arg-22 by Ala results in a more than 10-fold loss of the ability of AMP to inhibit the enzyme. These results are consistent wi th a role of Arg-22 in the preferential stabilization of the noncataly tic T state of the enzyme.