Glutamic acid residue 98 is critical for catalysis in pig kidney fructose-1,6-bisphosphatase.
Site-specific mutagenesis has been used to replace Glu-98 with flutamine in pig kidney fructose-1,6-bisphosphatase (Fru-1,6-P(2)ase) in order to evaluate the role of this residue in catalysis. The combination of lower k(cat) and higher K(m) resulted in an approximately 12,000-fold reduction in the catalytic efficiency of the Glu-98-->Gln enzyme when compared to the wild-type enzyme. The affinity of the enzyme for Mg(2+) and for the allosteric inhibitor AMP was altered only slightly; however, cooperativity in the binding of both of these effectors was eliminated. In addition, AMP could not fully inhibit the Glu-98-->Gln enzyme. These data suggest a critical role for the carboxylate of Glu-98 both as a general base in the reaction, and in the mechanism of allosteric inhibition of the enzyme by AMP.
Kelley
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N
Department of Chemistry, Boston College, Chestnut Hill, Massachusetts 02167, USA.
Giroux
E L
EL
Lu
G
G
Kantrowitz
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ER
D000595:Amino Acid Sequence; D000818:Animals; D001665:Binding Sites; D002384:Catalysis; D006597:Fructose-Bisphosphatase / Q000737:chemistry* / Q000378:metabolism*; D018698:Glutamic Acid; D005973:Glutamine; D007668:Kidney / Q000201:enzymology*; D007700:Kinetics; D016297:Mutagenesis, Site-Directed; D017354:Point Mutation; D011994:Recombinant Proteins / Q000737:chemistry / Q000378:metabolism; D013552:Swine
D011994:Recombinant Proteins; D005973:Glutamine; D018698:Glutamic Acid; D006597:Fructose-Bisphosphatase