Replacement of glutamic acid 29 with glutamine leads to a loss of cooperativity for AMP with porcine fructose-1,6-bisphosphatase.
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.
Chen
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M
Department of Biochemistry and Biophysics, Iowa State University, Ames 50011.
Chen
L
L
Fromm
H J
HJ
D000249:Adenosine Monophosphate / Q000378:metabolism*; D000818:Animals; D001483:Base Sequence; D001665:Binding Sites; D002942:Circular Dichroism; D017931:DNA Primers; D004789:Enzyme Activation; D006597:Fructose-Bisphosphatase / Q000037:antagonists & inhibitors / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism*; D005971:Glutamates / Q000378:metabolism*; D018698:Glutamic Acid; D005973:Glutamine / Q000378:metabolism*; D007700:Kinetics; D008099:Liver / Q000201:enzymology*; D008274:Magnesium / Q000494:pharmacology; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D013552:Swine
D017931:DNA Primers; D005971:Glutamates; D005973:Glutamine; D018698:Glutamic Acid; D000249:Adenosine Monophosphate; D006597:Fructose-Bisphosphatase; D008274:Magnesium