Hybrid tetramers of porcine liver fructose-1,6-bisphosphatase reveal multiple pathways of allosteric inhibition.
Fructose-1,6-bisphosphatase is a square planar tetramer of identical subunits, which exhibits cooperative allosteric inhibition of catalysis by AMP. Protocols for in vitro subunit exchange provide three of five possible hybrid tetramers of fructose-1,6-bisphosphatase in high purity. The two hybrid types with different subunits in the top and bottom halves of the tetramer co-purify. Hybrid tetramers, formed from subunits unable to bind AMP and subunits with wild-type properties, differ from the wild-type enzyme only in regard to their properties of AMP inhibition. Hybrid tetramers exhibit cooperative, potent, and complete (100%) AMP inhibition if at least one functional AMP binding site exists in the top and bottom halves of the tetramer. Furthermore, titrations of hybrid tetramers with AMP, monitored by a tryptophan reporter group, reveal cooperativity and fluorescence changes consistent with an R- to T-state transition, provided that again at least one functional AMP site exists in the top and bottom halves of the tetramer. In contrast, hybrid tetramers, which have functional AMP binding sites in only one half (top/bottom), exhibit an R- to T-state transition and complete AMP inhibition, but without cooperativity. Evidently, two pathways of allosteric inhibition of fructose-1,6-bisphosphatase are possible, only one of which is cooperative.
Nelson
Scott W
SW
Department of Biochemistry, Biophysics, and Molecular Biology, Iowa State University, Ames, Iowa 50011, USA.
Honzatko
Richard B
RB
Fromm
Herbert J
HJ
D000249:Adenosine Monophosphate / Q000378:metabolism / Q000494:pharmacology; D000494:Allosteric Regulation; D019943:Amino Acid Substitution; D000818:Animals; D001665:Binding Sites; D006597:Fructose-Bisphosphatase / Q000037:antagonists & inhibitors* / Q000737:chemistry; D007700:Kinetics; D008099:Liver / Q000201:enzymology*; D016297:Mutagenesis, Site-Directed; D021122:Protein Subunits; D011994:Recombinant Proteins / Q000037:antagonists & inhibitors / Q000737:chemistry; D013552:Swine
D021122:Protein Subunits; D011994:Recombinant Proteins; D000249:Adenosine Monophosphate; D006597:Fructose-Bisphosphatase