Role of Ser530, Arg292, and His662 in the allosteric behavior of rabbit muscle phosphofructokinase.
Fructose-2,6-bisphosphate (Fru-2,6-P(2)) is a potent allosteric activator of the ATP-dependent phosphofructokinase (PFK) in eukaryotes. Based on the sequence homology between rabbit muscle PFK and two bacterial PFKs and the crystal structures of the latter, Ser(530), Arg(292) and His(662) of the rabbit enzyme are implicated as binding sites for Fru-2,6-P(2). We report here the effects of three mutations, S530D, R292A, and H662A on the activation of rabbit muscle PFK by Fru-2,6-P(2). At pH 7.0 and the inhibitory concentrations of ATP, the native enzyme gives a classic sigmoidal response to changes in Fru-6-P concentration in the absence of Fru-2,6-P(2) and a nearly hyperbolic response in the presence of the activator. Under the same conditions, no activation was seen for S530D. On the other hand, H662A can be activated but requires a 10-fold or higher concentration of Fru-2,6-P(2). Limited activation was observed for mutant R292A. A model illustrating the sites for recognition of Fru-2,6-P(2) in rabbit muscle PFK as well as the mechanism of allosteric activation is proposed.
Chang
Simon H
SH
Department of Biological Sciences, Louisiana State University, Baton Rouge, Louisiana 70803, USA. schang@unix1.sncc.lsu.edu
Kemp
Robert G
RG
D000255:Adenosine Triphosphate / Q000378:metabolism; D000494:Allosteric Regulation / Q000187:drug effects / Q000502:physiology; D019943:Amino Acid Substitution; D000818:Animals; D001665:Binding Sites / Q000502:physiology; D004789:Enzyme Activation / Q000187:drug effects; D005635:Fructosediphosphates / Q000378:metabolism / Q000494:pharmacology; D005636:Fructosephosphates / Q000737:chemistry / Q000378:metabolism; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D025203:Phosphofructokinase-1, Muscle Type / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D011817:Rabbits; D017386:Sequence Homology, Amino Acid; D013329:Structure-Activity Relationship
D005635:Fructosediphosphates; D005636:Fructosephosphates; C027618:fructose-6-phosphate; C027652:fructose 2,6-diphosphate; D000255:Adenosine Triphosphate; D025203:Phosphofructokinase-1, Muscle Type