Replacement of glutamic acid 29 with glutamine leads to a loss of cooperativity for AMP with porcine fructose-1,6-bisphosphatase.

Abstract

Mutations in the AMP binding site of porcine fructose-1,6-bisphosphatase were carried out by site-specific mutagenesis based on the crystal structure of the enzyme (Ke, H., Zhang, Y., and Lipscomb, W.L. (1990) Proc. Natl. Acad. Sci. U.S.A. 87, 5243-5247). The mutant and wild-type enzymes were characterized by SDS-polyacrylamide gel electrophoresis, circular dichroism spectrometry, and initial rate kinetics. One of the mutant forms of fructose-1,6-bisphosphatase, Glu-29-->Gln, is ligated to the phosphoryl moiety of AMP, a potent inhibitor of the reaction, whereas the other mutant, Thr-31-->Val, is associated with the purine base of AMP. No discernible alteration in structure as measured by circular dichroism spectrometry was noted for the mutants relative to the wild-type enzyme. As expected, major changes in kinetic parameters between the mutants and the wild-type enzyme were associated with inhibition by AMP. AMP, a competitive inhibitor with respect to Mg2+ in the fructose-1,6-bisphosphatase reaction, exhibits cooperativity in the case of the wild-type and the mutant Thr-31-->Val enzymes with a Hill coefficient of 2.0. On the other hand, cooperativity is completely lost in the case of Glu-29-->Gln fructose-1,6-bisphosphatase.