Two Binding Sites Involved in Bacterial ADP-glucose Pyrophosphorylases Activation

M.D. ASENCIÓN DIEZ; M. ALEANZI; M.A. BALLICORA; A.A. IGLESIAS

Mendoza

Congreso; XLVIII Reunión Anual de la SAIB; 2012

SAIB

Bacterial glycogen synthesis is allosterically regulated at the levelof ADPGlc pyrophosphorylase (ADPGlcPPase) by intracellularmetabolites. In the enzyme is activatedby pyruvate (Pyr) and fructose-6P (F6P) while inby fructose-1,6-bisP (FBP). Previously, the -ADPGlcPPaseactivation kinetics were extensively characterized and two aminoacid residues were identified as critical for allosteric response sincemutants Q74A and W113A are insensitive to FBP. Crosstalkanalysis between Pyr and F6P activation in wild-type -ADPGlcPPase, together with studies on the behavior of Q67A andW106A mutant enzymes (homologous to mutants) wereconducted. Noteworthy, W106A lost sensitivity to F6P althoughwas still activated by Pyr. Results allowed us to propose two bindingsites involved in allosteric regulation. Also, kinetic properties ofchimeric proteins made by switching the N-term and C-termportions of and enzymes support this hypothesis.We furtherstudied -ADPGlcPPase activation, determining that Pyr has asynergistic role in FBP activation. Also, combination of Pyr withanother effector in the enzyme addedsustain to the two sites model for regulation in bacterialADPGlcPPases. One of these sites is related with Pyr, behaving asfine modulator that governs activity and/or sensitivity to othereffectors.