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; 28 346 Mr) 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 Tm = 57 degrees C and an unfolding enthalpy of approximately 140 kcal/mol. In contrast, monomeric EIN expressed in a wild-type strain (pts+) had two endotherms with Tm congruent with 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 degrees C (without changes in CD: approximately 58% alpha-helices). Thermal conversion to a more stable form was correlated with dephosphorylation of EIN by mass spectral analysis. Dephospho-enzyme I (monomer right arrow over left arrow dimer) exhibited endotherms for C- and N-terminal domain unfolding with Tm = 41 and 54 degrees C, respectively. Thermal unfolding of the C-terminal domain occurred over a broad temperature range ( approximately 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 Tm = 54 to approximately 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, HP(r).
Nosworthy
N J
NJ
Section on Protein Chemistry, Laboratory of Biochemistry, National Heart, Lung, and Blood Institute, Bethesda, Maryland 20892, USA.
Peterkofsky
A
A
König
S
S
Seok
Y J
YJ
Szczepanowski
R H
RH
Ginsburg
A
A
D002152:Calorimetry, Differential Scanning; D002942:Circular Dichroism; D004926:Escherichia coli / Q000201:enzymology*; D008027:Light; D010731:Phosphoenolpyruvate Sugar Phosphotransferase System / Q000737:chemistry / Q000378:metabolism*; D010766:Phosphorylation; D017852:Phosphotransferases (Nitrogenous Group Acceptor) / Q000737:chemistry / Q000378:metabolism*; D017510:Protein Folding; D017434:Protein Structure, Tertiary; D012542:Scattering, Radiation; D013050:Spectrometry, Fluorescence; D013816:Thermodynamics
D010731:Phosphoenolpyruvate Sugar Phosphotransferase System; C031629:phosphoenolpyruvate-glucose phosphotransferase; D017852:Phosphotransferases (Nitrogenous Group Acceptor); C022360:phosphoenolpyruvate-protein phosphotransferase