Campus Units
Biochemistry, Biophysics and Molecular Biology
Document Type
Article
Publication Version
Published Version
Publication Date
9-2000
Journal or Book Title
Journal of Biological Chemistry
Volume
275
Issue
39
First Page
29986
Last Page
29992
DOI
10.1074/jbc.M000473200
Abstract
Loop 52–72 of porcine fructose-1,6-bisphosphatase may play a central role in the mechanism of catalysis and allosteric inhibition by AMP. The loop pivots between different conformational states about a hinge located at residues 50 and 51. The insertion of proline separately at positions 50 and 51 reduces k cat by up to 3-fold, with no effect on the K m for fructose 1,6-bisphosphate. TheK a for Mg2+ in the Lys50→ Pro mutant increases ∼15-fold, whereas that for the Ala51 → Pro mutant is unchanged. Although these mutants retain wild-type binding affinity for AMP and the fluorescent AMP analog 2′(3′)-O-(trinitrophenyl)adenosine 5′-monophosphate, the K i for AMP increases 8000- and 280-fold in the position 50 and 51 mutants, respectively. In fact, the mutation Lys50 → Pro changes the mechanism of AMP inhibition with respect to Mg2+ from competitive to noncompetitive and abolishes K+ activation. The K i for fructose 2,6-bisphosphate increases ∼20- and 30-fold in the Lys50 → Pro and Ala51 → Pro mutants, respectively. Fluorescence from a tryptophan introduced by the mutation of Tyr57suggests an altered conformational state for Loop 52–72 due to the proline at position 50. Evidently, the Pro50 mutant binds AMP with high affinity at the allosteric site, but the mechanism of allosteric regulation of catalysis has been disabled.
Copyright Owner
American Society for Biochemistry and Molecular Biology
Copyright Date
2000
Language
en
File Format
application/pdf
Recommended Citation
Nelson, Scott W.; Choe, Jun-yong; Honzatko, Richard B.; and Fromm, Herbert J., "Mutations in the Hinge of a Dynamic Loop Broadly Influence Functional Properties of Fructose-1,6-bisphosphatase" (2000). Biochemistry, Biophysics and Molecular Biology Publications. 79.
https://lib.dr.iastate.edu/bbmb_ag_pubs/79
Comments
This article is from Journal of Biological Chemistry 275 (2000): 29986, doi: 10.1074/jbc.M000473200. Posted with permission.