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.

Comments

This article is from Journal of Biological Chemistry 275 (2000): 29986, doi: 10.1074/jbc.M000473200. Posted with permission.

Copyright Owner

American Society for Biochemistry and Molecular Biology

Language

en

File Format

application/pdf

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