Understanding Allosteric Regulation of ADPglucose Pyrophosphorylase from Escherichia coli

M.C. ESPER, M.C. ALEANZI, M.A. BALLICORA, A.A. IGLESIAS

Mar del Plata, Buenos Aires, Argentina

Congreso; XLIII Reunión Anual de SAIB; 2007

SAIB

EN-P19.UNDERSTANDING ALLOSTERIC REGULATION OFADPGLUCOSE PYROPHOSPHORYLASE FROMESCHERICHIACOLIEsper MC, Aleanzi MC, Ballicora MA, Iglesias AA.Lab Enz Mol, FBCB, UNL, Santa Fe, Argentina and *DeptChemistry, Loyola University at Chicago, USA. E-mail:mesper@fbcb.unl.edu.arADPglucose pyrophosphorylase (ADPGlcPPase) from E. coli isallosterically activated by fructose-1,6-bisphosphate (FBP), andinhibited by AMP. To elucidate structure to function relationships,we constructed the double mutant enzymes: Q74A/G336D,W113A/G336D, N- 15/Q74A and N- 15/W113A. Structuralcharacteristics of these mutants combine changes in protein domainsidentified with regulatory properties. Single mutants Q74A andW113Ahave similar specific activity than the wild type enzyme, butare insensitive to FBP activation. Mutants G336D and N- 15 arepre-activated enzymes, as they exhibit specific activity, in theabsence of activator, comparable to that of the wild type in thepresence of FBP. The introduction of the second mutation (W113Aor Q74A) decreased sensitivity to FBP activation and AMPinhibition of the pre-activated mutants, suggesting that theseresidues are critical for the conformational change induced by theactivator. Also, introduction of the second mutation decreased thespecific activity of N- 15 in the absence of FBP, but scarcelyaffected G336D kinetic behavior. It is suggested that the activatedconformation arises from two different changes in protein structureat the N- and C-term domains, respectively. Results reinforce theidea that AMP inhibition in the mutant proteins, as in the wild typeenzyme, mainly modifies the activation produced by FBP.