AMP inhibition of pig kidney fructose-1,6-bisphosphatase

作者：Kelley Loughnane N; Kantrowitz ER^*

来源：Biochimica et Biophysica Acta - Protein Structure and Molecular Enzymology, 2001, 1548(1): 66-71.

DOI：10.1016/S0167-4838(01)00218-7

• allosteric regulation
• site-specific mutagenesis
• phosphate hydrolysis

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 Mg2+ 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 wildtype enzyme. The K112Q enzyme also showed a 10-fold reduction in affinity for Mg2+. Although the allosteric site is approximately 28 Angstrom 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.

• 出版日期2001-7-9