Mutations in the hinge of a dynamic loop broadly influence functional properties of fructose-1,6-bisphosphatase.
Loop 52-72 of porcine fructose-1,6-bisphosphatase may play a central role in the mechanism of catalysis and allosteric inhibition by AMP. The loop pivots between different conformational states about a hinge located at residues 50 and 51. The insertion of proline separately at positions 50 and 51 reduces k(cat) by up to 3-fold, with no effect on the K(m) for fructose 1,6-bisphosphate. The K(a) for Mg(2+) in the Lys(50) --> Pro mutant increases approximately 15-fold, whereas that for the Ala(51) --> Pro mutant is unchanged. Although these mutants retain wild-type binding affinity for AMP and the fluorescent AMP analog 2'(3')-O-(trinitrophenyl)adenosine 5'-monophosphate, the K(i) for AMP increases 8000- and 280-fold in the position 50 and 51 mutants, respectively. In fact, the mutation Lys(50) --> Pro changes the mechanism of AMP inhibition with respect to Mg(2+) from competitive to noncompetitive and abolishes K(+) activation. The K(i) for fructose 2,6-bisphosphate increases approximately 20- and 30-fold in the Lys(50) --> Pro and Ala(51) --> Pro mutants, respectively. Fluorescence from a tryptophan introduced by the mutation of Tyr(57) suggests an altered conformational state for Loop 52-72 due to the proline at position 50. Evidently, the Pro(50) mutant binds AMP with high affinity at the allosteric site, but the mechanism of allosteric regulation of catalysis has been disabled.
Nelson
S W
SW
Department of Biochemistry, Biophysics, and Molecular Biology, Iowa State University, Ames, Iowa 50011, USA.
Choe
J Y
JY
Honzatko
R B
RB
Fromm
H J
HJ
D000249:Adenosine Monophosphate / Q000031:analogs & derivatives / Q000378:metabolism; D000409:Alanine / Q000235:genetics; D000494:Allosteric Regulation / Q000235:genetics; D000818:Animals; D020134:Catalytic Domain / Q000235:genetics; D006597:Fructose-Bisphosphatase / Q000037:antagonists & inhibitors / Q000235:genetics / Q000378:metabolism*; D007700:Kinetics; D008239:Lysine / Q000235:genetics; D008956:Models, Chemical; D008958:Models, Molecular; D016297:Mutagenesis, Site-Directed; D009154:Mutation; D011392:Proline / Q000235:genetics; D013552:Swine
D000249:Adenosine Monophosphate; C036744:2',3'-O (2,4,6-trinitrocyclohexadienylidine)adenosine 5'-monophosphate; D011392:Proline; D006597:Fructose-Bisphosphatase; D008239:Lysine; D000409:Alanine