Mutation of arginine 276 to methionine changes Mg2+ cooperativity and the kinetic mechanism of fructose-1,6-bisphosphatase.
Arginine 276 of porcine liver fructose-1,6-bisphosphatase (FBPase) was mutated to methionine by site-directed mutagenesis on the basis of the crystal structure of the enzyme [Zhang, Y., Liang, J.-Y., Huang, S., Ke, H., & Lipscomb, W.N. (1993) Biochemistry 32, 1844-1857]. The mutant and wild-type forms of the enzyme were purified to homogeneity and characterized by circular dichroism spectrometry (CD) and initial-rate kinetics. There were no discernible differences between the secondary structures of the wild-type and the mutant enzymes on the basis of the CD data. Replacement of Arg 276 with methionine caused a significant decrease in the enzyme's activity. The kcat for the mutant enzyme was only about 0.67% of that of the wild-type enzyme. Most importantly, the mutation caused the total loss of cooperativity for Mg2+ and changed the kinetic mechanism to one in which the substrate adds to FBPase before Mg2+ and in which all steps equilibrate rapidly relative to the conversion of the ternary complex of enzyme, substrate, and Mg2+ to products. The Ka for Mg2+ increased by only about 5-fold relative to that of the wild-type enzyme. The mutation did not change the Ki for AMP or the Hill coefficient of this allosteric inhibitor. The Ki for fructose 2,6-bisphosphate was increased by 16-fold compared with that of the wild-type enzyme. The Km for fructose 1,6-bisphosphate was similar to that of the wild-type enzyme. It is concluded that Arg 276 is critical for activity and Mg2+ cooperativity with FBPase and it determines the enzyme's kinetic mechanism.
Zhang
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Department of Biochemistry and Biophysics, Iowa State University, Ames 50011, USA.
Fromm
H J
HJ
D000249:Adenosine Monophosphate / Q000494:pharmacology; D000818:Animals; D001120:Arginine; D001483:Base Sequence; D004591:Electrophoresis, Polyacrylamide Gel; D006597:Fructose-Bisphosphatase / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D005635:Fructosediphosphates / Q000494:pharmacology; D007700:Kinetics; D008099:Liver / Q000201:enzymology; D008274:Magnesium / Q000494:pharmacology*; D008715:Methionine; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D017433:Protein Structure, Secondary; D013329:Structure-Activity Relationship; D013552:Swine
D005635:Fructosediphosphates; D000249:Adenosine Monophosphate; C027652:fructose 2,6-diphosphate; D001120:Arginine; D008715:Methionine; D006597:Fructose-Bisphosphatase; D008274:Magnesium