Directed mutations in the poorly defined region of porcine liver fructose-1,6-bisphosphatase significantly affect catalysis and the mechanism of AMP inhibition.
Asn64, Asp68, Lys71, Lys72, and Asp74 of porcine liver fructose-1, 6-bisphosphatase (FBPase) are conserved residues and part of a loop for which no electron density has been observed in crystal structures. Yet mutations of the above dramatically affect catalytic rates and/or AMP inhibition. The Asp74 --> Ala and Asp74 --> Asn mutant enzymes exhibited 50,000- and 2,000-fold reductions, respectively, in kcat relative to wild-type FBPase. The pH optimum for the catalytic activity of the Asp74 --> Glu, Asp68 --> Glu, Asn64 --> Gln, and Asn64 --> Ala mutant enzymes shifted from pH 7.0 (wild-type enzyme) to pH 8.5, whereas the Lys71 --> Ala mutant and Lys71,72 --> Met double mutant had optimum activity at pH 7.5. Mg2+ cooperativity, Km for fructose 1,6-bisphosphate, and Ki for fructose 2,6-bisphosphate were comparable for the mutant and wild-type enzymes. Nevertheless, for the Asp74 --> Glu, Asp68 --> Glu, Asn64 --> Gln, and Asn64 --> Ala mutants, the binding affinity for Mg2+ decreased by 40-125-fold relative to the wild-type enzyme. In addition, the Asp74 --> Glu and Asn64 --> Ala mutants exhibited no AMP cooperativity, and the kinetic mechanism of AMP inhibition with respect to Mg2+ was changed from competitive to noncompetitive. The double mutation Lys71,72 --> Met increased Ki for AMP by 175-fold and increased Mg2+ affinity by 2-fold relative to wild-type FBPase. The results reported here strongly suggest that loop 51-72 is important for catalytic activity and the mechanism of allosteric inhibition of FBPase by AMP.
Kurbanov
F T
FT
Department of Biochemistry and Biophysics, Iowa State University, Ames, Iowa 50011, USA.
Choe
J Y
JY
Honzatko
R B
RB
Fromm
H J
HJ
D000249:Adenosine Monophosphate / Q000494:pharmacology*; D000494:Allosteric Regulation; D019943:Amino Acid Substitution; D000818:Animals; D001483:Base Sequence; D002384:Catalysis; D017931:DNA Primers; D006597:Fructose-Bisphosphatase / Q000037:antagonists & inhibitors / Q000378:metabolism*; D007700:Kinetics; D016297:Mutagenesis, Site-Directed; D013552:Swine
D017931:DNA Primers; D000249:Adenosine Monophosphate; D006597:Fructose-Bisphosphatase