Major changes in the kinetic mechanism of AMP inhibition and AMP cooperativity attend the mutation of Arg49 in fructose-1,6-bisphosphatase.
The significance of subunit interface residues Arg49 and Lys50 in the function of porcine liver fructose-1,6-bisphosphatase was explored by site-directed mutagenesis, initial rate kinetics, and circular dichroism spectroscopy. The Lys50 --> Met mutant had kinetic properties similar to the wild-type enzyme but was more thermostable. Mutants Arg49 --> Leu, Arg49 --> Asp, Arg49 --> Cys were less thermostable than the wild-type enzyme yet exhibited wild-type values for kcat and Km. The Ki for the competitive inhibitor fructose 2,6-bisphosphate increased 3- and 5-fold in Arg49 --> Leu and Arg49 --> Asp, respectively. The Ka for Mg2+ increased 4-8-fold for the Arg49 mutants, with no alteration in the cooperativity of Mg2+ binding. Position 49 mutants had 4-10-fold lower AMP affinity. Most significantly, the mechanism of AMP inhibition with respect to fructose 1,6-bisphosphate changed from noncompetitive (wild-type enzyme) to competitive (Arg49 --> Leu and Arg49 --> Asp mutants) and to uncompetitive (Arg49 --> Cys mutant). In addition, AMP cooperativity was absent in the Arg49 mutants. The R and T-state circular dichroism spectra of the position 49 mutants were identical and superimposable on only the R-state spectrum of the wild-type enzyme. Changes from noncompetitive to competitive inhibition by AMP can be accommodated within the framework of a steady-state Random Bi Bi mechanism. The appearance of uncompetitive inhibition, however, suggests that a more complex mechanism may be necessary to account for the kinetic properties of the enzyme.
Shyur
L F
LF
Department of Biochemistry and Biophysics, Iowa State University, Ames, Iowa 50011, USA.
Poland
B W
BW
Honzatko
R B
RB
Fromm
H J
HJ
D000249:Adenosine Monophosphate / Q000037:antagonists & inhibitors* / Q000378:metabolism*; D001120:Arginine / Q000235:genetics / Q000378:metabolism*; D002942:Circular Dichroism; D006597:Fructose-Bisphosphatase / Q000235:genetics / Q000378:metabolism*; D007700:Kinetics; D016296:Mutagenesis; D013696:Temperature
D000249:Adenosine Monophosphate; D001120:Arginine; D006597:Fructose-Bisphosphatase