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Crystal structures of the active site mutant (Arg‐243 → Ala) in the T and R allosteric states of pig kidney fructose‐1,6‐bisphosphatase expressed in escherichia coli
Author(s) -
Stec Boguslaw,
Abraham Reimar,
Giroux Eugene,
Kantrowitz Evan R.
Publication year - 1996
Publication title -
protein science
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 3.353
H-Index - 175
eISSN - 1469-896X
pISSN - 0961-8368
DOI - 10.1002/pro.5560050810
Subject(s) - allosteric regulation , fructose 1,6 bisphosphatase , active site , enzyme , binding site , fructose , mutant , chemistry , stereochemistry , conformational change , biochemistry , gene
Abstract The active site of pig kidney fructose‐l,6‐bisphosphatase (EC 3.1.3.11) is shared between subunits, Arg‐243 of one chain interacting with fructose‐1,6‐bisphosphate or fructose‐2,6‐bisphosphate in the active site of an adjacent chain. In this study, we present the X‐ray structures of the mutant version of the enzyme with Arg‐243 replaced by alanine, crystallized in both T and R allosteric states. Kinetic characteristics of the altered enzyme showed the magnesium binding and inhibition by AMP differed slightly; affinity for the substrate fructose‐1,6‐bisphosphate was reduced 10‐fold and affinity for the inhibitor fructose‐2,6‐bisphosphate was reduced 1,000‐fold (Giroux E, Williams MK, Kantrowitz ER, 1994, J Biol Chem 269 :31404–31409). The X‐ray structures show no major changes in the organization of the active site compared with wild‐type enzyme, and the structures confirm predictions of molecular dynamics simulations involving Lys‐269 and Lys‐274. Comparison of two independent models of the T form structures have revealed small but significant changes in the conformation of the bound AMP molecules and small reorganization of the active site correlated with the presence of the inhibitor. The differences in kinetic properties of the mutant enzyme indicate the key importance of Arg‐243 in the function of fructose‐1,6‐bisphosphatase. Calculations using the X‐ray structures of the Arg‐243 Ala enzyme suggest that the role of Arg‐243 in the wild‐type enzyme is predominantly electrostatic in nature.