ABSTRACT
AMP is an allosteric inhibitor of human muscle and liver fructose-1,6-bisphosphatase (FBPase). Despite strong similarity of the nucleotide binding domains, the muscle enzyme is inhibited by AMP approximately 35 times stronger than liver FBPase: I0.5 for muscle and for liver FBPase are 0.14 microM and 4.8 microM, respectively. Chimeric human muscle (L50M288) and chimeric human liver enzymes (M50L288), in which the N-terminal residues (1-50) were derived from the human liver and human muscle FBPases, respectively, were inhibited by AMP 2-3 times stronger than the wild-type liver enzyme. An amino acid exchange within the N-terminal region of the muscle enzyme towards liver FBPase (Lys20-->Glu) resulted in 13-fold increased I0.5 values compared to the wild-type muscle enzyme. However, the opposite exchanges in the liver enzyme (Glu20-->Lys and double mutation Glu19-->Asp/Glu20-->Lys) did not change the sensitivity for AMP inhibition of the liver mutant (I0.5 value of 4.9 microM). The decrease of sensitivity for AMP of the muscle mutant Lys20-->Glu, as well as the lack of changes in the inhibition by AMP of liver mutants Glu20-->Lys and Glu19-->Asp/Glu20-->Lys, suggest a different mechanism of AMP binding to the muscle and liver enzyme.
Subject(s)
Adenosine Monophosphate/pharmacology , Enzyme Inhibitors/pharmacology , Fructose-Bisphosphatase/antagonists & inhibitors , Fructose-Bisphosphatase/genetics , Liver/enzymology , Muscle, Skeletal/enzymology , Mutation/physiology , Recombinant Fusion Proteins/antagonists & inhibitors , Recombinant Fusion Proteins/genetics , Binding Sites/drug effects , DNA Primers , Escherichia coli/metabolism , Fructose-Bisphosphatase/biosynthesis , Humans , Isoenzymes/antagonists & inhibitors , Isoenzymes/genetics , Kinetics , Magnesium/pharmacology , Models, Molecular , Molecular Conformation , Mutagenesis, Site-Directed , NAD/pharmacologyABSTRACT
A comparison of the amino acid sequences of the liver and muscle fructose-1,6-bisphosphatase (FbPase) isoforms in primates and rodents suggested an ancient duplication event leading to the corresponding genes. We investigated the presence of both genes in the rabbit (order lagomorphs) and in species belonging to further distantly related metazoan taxa. By an analysis of the available complete genomes and proteomes of the nematode Caenorhabditis elegans and of Drosophila melanogaster only one sequence homologous to known FbPases was found in each species. The corresponding mRNAs were characterized by cDNA sequencing. We then carried out reverse transcription-polymerase chain reactions to amplify central fragments of the FbPase cDNAs from liver and muscle of Gallus gallus, Xenopus laevis, and Esox lucius, respectively. Their sequencing revealed that (i) the livers of chicken, frog, and fish contain mRNAs which are closely related to mammalian liver FbPase mRNAs, (ii) chicken muscle contains an mRNA which is most homologous to mammalian muscle FbPase mRNAs, (iii) frog muscle contains both a liver-type and a muscle-type FbPase mRNA, while (iv) in fish muscle no FbPase mRNA could be detected by our approach despite the doubtless presence of the enzyme in this organ. An alignment of the partial amino acid sequences of the different FbPases showed that the residues that are thought to be in contact with the substrate, fructose-2,6-bisphosphate, and Mg(2+) are totally conserved, while some amino acids having contact with adenosine monophosphate were found to vary among several species. The question of what might be the advantage of having more than one gene coding for FbPase per haploid genome is discussed.
