Affinity labeling of the allosteric site of fructose 1,6-bisphosphatase with an AMP analog.
D-Fructose 1,6-bisphosphatase [EC 3.1.3.11, FBPase] is one of the key enzymes in glyconeogenesis and its activity is controlled by various effectors such as substrate, AMP and ATP. To analyze this complex regulation system, we tried an affinity labeling of FBPase with an AMP derivative, since AMP is a potent allosteric inhibitor of this enzyme. The results obtained are as follows. 1. To determine the functional groups which are essential for AMP as an inhibitor, inhibitory activities of some AMP derivatives were examined. These derivatives modified at the purine ring or phosphate group lost the activity while one modified at the ribose ring retained the ability to inhibit FBPase. This shows that an affinity labeling reagent should be an AMP derivative in which the ribose ring is modified. 2. 2',3'-Dialdehyde AMP (dial-AMP) was prepared by periodate oxidation of AMP and was reacted with FBPase. Under appropriate conditions, 1 mol of the reagent was incorporated per mol of enzyme subunit with a concomitant loss of enzyme activity. The reaction was prevented by the presence of AMP but not of ATP. The heat-stability, the kinetic parameters and the UV-absorption spectrum of the modified enzyme were all the same as those of native FBPase in the presence of AMP. Thus it was concluded that the allosteric AMP site in FBPase was modified specifically.
Sakai
K
K
Department of Biochemistry, Faculty of Medicine, University of Tokyo.
Kawashima
S
S
Suzuki
K
K
Imahori
K
K
D000249:Adenosine Monophosphate / Q000031:analogs & derivatives* / Q000138:chemical synthesis / Q000494:pharmacology; D000345:Affinity Labels / Q000494:pharmacology*; D000495:Allosteric Site; D000818:Animals; D002645:Chickens; D002942:Circular Dichroism; D004228:Dithionitrobenzoic Acid / Q000494:pharmacology; D006597:Fructose-Bisphosphatase / Q000378:metabolism*; D007700:Kinetics; D008099:Liver / Q000201:enzymology; D011487:Protein Conformation; D013438:Sulfhydryl Compounds / Q000032:analysis
D000345:Affinity Labels; D013438:Sulfhydryl Compounds; C041129:adenosine 5'-monophosphate 2',3'-dialdehyde; D000249:Adenosine Monophosphate; D004228:Dithionitrobenzoic Acid; D006597:Fructose-Bisphosphatase