Phosphorylation Destabilizes The Amino-Terminal Domain Of Enzyme I Of The Escherichia Coli Phosphoenolpyruvate : Sugar Phosphotransferase System

Thermal stabilities of enzyme I (63 562 M-r subunit), in the Escherichia coli phosphoenolpyruvate (PEP),sugar phosphotransferase system (PTS), and a cloned amino-terminal domain of enzyme I (EIN; 28 34-6 M,) were investigated by differential scanning calorimetry (DSC) and far-UV circular dichroism (CD) at PH 7.5. EIN expressed in a Delta pts E. coli strain showed a single, reversible, two-state transition with T-m = 57 degrees C and an unfolding enthalpy of similar to 140 kcal/mol, In contrast, monomeric EIN expressed in a wild-type strain (pts(+)) had two endotherms with T-m congruent to 50 and 57 degrees C and overall Delta H = 140 kcal/mol and was converted completely to the more stable form after five DSC scans from 10 to 75 DC (without changes in CD: similar to 58% alpha-helices). Thermal conversion to a more stable form was correlated with dephosphorylation of EIN by mass spectral analysis. Dephospho-enzyme I (monomer reversible arrow dimer) exhibited endotherms for C-and N-terminal domain unfolding with T-m = 41 and 54 degrees C, respectively. Thermal unfolding of the C-terminal domain occurred over a broad temperature range (similar to 30-50 degrees C), was scan rate-and concentration-dependent, coincident with a Light scattering decrease and Trp residue exposure, and independent of phosphorylation. Reversible thermal unfolding of the nonphosphorylated N-terminal domain was more cooperative, occurring from 50 to 60 degrees C. DSC of partially phosphorylated enzyme I indicated that the amino-terminal domain was destabilized by phosphorylation (from T-m = 54 to similar to 48 degrees C). A decrease in conformational stability of the amino-terminal domain of enzyme I produced by phosphorylation of the active-site His 189 has the physiological consequence of promoting phosphotransfer to the phosphocarrier protein, HPr.