Shared active sites of fructose-1,6-bisphosphatase. Arginine 243 mediates substrate binding and fructose 2,6-bisphosphate inhibition.
The active site of pig kidney fructose-1,6-bisphosphatase (EC 3.1.3.11) is shared between subunits, Arg-243 of one chain interacting with fructose-1,6-bisphosphate or fructose-2,6-bisphosphate in the active site of an adjacent chain. In this study, Arg-243 was replaced by alanine using techniques of site-specific mutagenesis and the cloned pig kidney enzyme expressed in Escherichia coli. Compared with wild-type enzyme, kinetic parameters of the altered enzyme characterizing catalytic efficiency, magnesium binding, and inhibition by AMP differed but by less than an order of magnitude; affinity for substrate fructose 1,6-bisphosphate was 10-fold poorer, and affinity for inhibitor fructose 2,6-bisphosphate was 1000-fold poorer. Molecular dynamics simulations were undertaken to determine possible alterations in active sites of the enzyme due to replacement of Arg-243 by Ala and suggested that in the mutant enzyme loss of one cationic group leads to reorganization of the active site especially involving lysine residues 269 and 274. The differences in properties of the mutant enzyme indicate the key importance of Arg-243 in the function of fructose-1,6-bisphosphatase and confirm on a functional basis the shared active site in this important metabolic enzyme.
Giroux
E
E
Department of Chemistry, Merkert Chemistry Center, Boston College, Chestnut Hill, Massachusetts 02167.
Williams
M K
MK
Kantrowitz
E R
ER
D000249:Adenosine Monophosphate / Q000378:metabolism; D000818:Animals; D001120:Arginine / Q000737:chemistry; D001665:Binding Sites; D002384:Catalysis; D006597:Fructose-Bisphosphatase / Q000037:antagonists & inhibitors / Q000737:chemistry* / Q000378:metabolism; D005635:Fructosediphosphates / Q000378:metabolism / Q000494:pharmacology; D066298:In Vitro Techniques; D007700:Kinetics; D008274:Magnesium / Q000378:metabolism; D008958:Models, Molecular; D016297:Mutagenesis, Site-Directed; D013329:Structure-Activity Relationship; D013552:Swine
D005635:Fructosediphosphates; D000249:Adenosine Monophosphate; C027652:fructose 2,6-diphosphate; D001120:Arginine; D006597:Fructose-Bisphosphatase; D008274:Magnesium; C029063:fructose-1,6-diphosphate