AMP inhibition of pig kidney fructose-1,6-bisphosphatase.
Lys-112 and Tyr-113 in pig kidney fructose-1,6-bisphosphatase (FBPase) make direct interactions with AMP in the allosteric binding site. Both residues interact with the phosphate moiety of AMP while Tyr-113 also interacts with the 3'-hydroxyl of the ribose ring. The role of these two residues in AMP binding and allosteric inhibition was investigated. Site-specific mutagenesis was used to convert Lys-112 to glutamine (K112Q) and Tyr-113 to phenylalanine (Y113F). These amino acid substitutions result in small alterations in k(cat) and increases in K(m). However, both the K112Q and Y113F enzymes show alterations in Mg(2+) affinity and dramatic reductions in AMP affinity. For both mutant enzymes, the AMP concentration required to reduced the enzyme activity by one-half, [AMP](0.5), was increased more than a 1000-fold as compared to the wild-type enzyme. The K112Q enzyme also showed a 10-fold reduction in affinity for Mg(2+). Although the allosteric site is approximately 28 A from the metal binding sites, which comprise part of the active site, these site-specific mutations in the AMP site influence metal binding and suggest a direct connection between the allosteric and the active sites.
Kelley-Loughnane
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Department of Chemistry, Boston College, Merkert Chemistry Center, Chestnut Hill, MA 02467, USA.
Kantrowitz
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D000249:Adenosine Monophosphate / Q000737:chemistry / Q000494:pharmacology*; D000495:Allosteric Site; D000818:Animals; D001665:Binding Sites; D002413:Cations, Divalent; D006597:Fructose-Bisphosphatase / Q000037:antagonists & inhibitors* / Q000737:chemistry / Q000235:genetics; D007668:Kidney / Q000187:drug effects* / Q000201:enzymology; D007700:Kinetics; D008274:Magnesium / Q000737:chemistry; D008958:Models, Molecular; D016297:Mutagenesis, Site-Directed; D009154:Mutation; D011485:Protein Binding; D013552:Swine
D002413:Cations, Divalent; D000249:Adenosine Monophosphate; D006597:Fructose-Bisphosphatase; D008274:Magnesium