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  • Engineering the allosteric properties of archaeal non-phosphorylating glyceraldehyde-3-phosphate dehydrogenases.

Engineering the allosteric properties of archaeal non-phosphorylating glyceraldehyde-3-phosphate dehydrogenases.

Biochimica et biophysica acta (2014-02-05)
Fumiaki Ito, Masayuki Miyake, Shinya Fushinobu, Shugo Nakamura, Kentaro Shimizu, Takayoshi Wakagi
ABSTRACT

The archaeal non-phosphorylating glyceraldehyde-3-phosphate dehydrogenase (GAPN, EC 1.2.1.9) is a highly allosteric enzyme activated by glucose 1-phosphate (Glc1P). Recent kinetic analyses of two GAPN homologs from Sulfolobales show different allosteric behaviors toward the substrate glyceraldehyde-3-phosphate (GAP) and the allosteric effector Glc1P. In GAPN from Sulfolobus tokodaii (Sto-GAPN), Glc1P-induced activation follows an increase in affinity for GAP rather than an increase in maximum velocity, whereas in GAPN from Sulfolobus solfataricus (Sso-GAPN), Glc1P-induced activation follows an increase in maximum velocity rather than in affinity for GAP. To explore the molecular basis of this difference between Sto-GAPN and Sso-GAPN, we generated 14 mutants and 2 chimeras. The analyses of chimeric GAPNs generated from regions of Sto-GAPN and Sso-GAPN indicated that a 57-residue module located in the subunit interface was clearly involved in their allosteric behavior. Among the point mutations in this modular region, the Y139R variant of Sto-GAPN no longer displayed a sigmoidal K-type-like allostery, but instead had apparent V-type allostery similar to that of Sso-GAPN, suggesting that the residue located in the center of the homotetramer critically contributes to the allosteric behavior.

MATERIALS
Product Number
Brand
Product Description

Sigma-Aldrich
α-D-Glucose 1-phosphate disodium salt hydrate, ≥97% (Enzymatic Purity, anhydrous)
Sigma-Aldrich
α-D-Glucose 1-phosphate dipotassium salt hydrate, ≥97% (HPLC)
Sigma-Aldrich
α-D-Glucose 1-phosphate dipotassium salt hydrate, ≥99% (HPLC), BioXtra