The widespread bacterial phosphoenolpyruvate (PEP) sugar phosphotransferase system (PTS) is capable of carrying out the coupled translocation and phosphorylation of numerous sugars. The system is composed of two cytoplasmic proteins (enzyme I and HPr) that are used for all sugars and sugar-specific proteins known as enzyme II. We have been investigating structure/function relationships of E. coli enzyme I (MW=63,500) and its N-terminal domain (EIN, MW = 28,346), which is inactive in autophosphorylation but is phosphorylated at His 189 by transfer from phospho-HPr. Previously, we found that phosphorylation of the active-site His 189 destabilizes the amino terminal domain of enzyme I, as shown by a 7 deg C decrease in the Tm of the two-state thermal unfolding transition at pH 7.5. A decrease in the conformational stability of the amino terminal domain by phosphorylation of His 189 promotes phosphotransfer to HPr. In order to investigate the effects of phosphorylation on the stability of enzyme I further, we have substituted Ala and Glu for His 189 to produce H189A and H189E mutants of both EIN and the full length enzyme I. These proteins have been expressed and purified. Differential scanning calorimetry (DSC) and temperature-induced changes in circular dichroism (CD) at 222 nm for wild-type (wt: H189-) and mutant EIN proteins have shown two-state unfolding for all three proteins in 10 mM K-phosphate, pH 7.5 buffer with the following parameters: Tm = 57 deg C ([Delta H] = 140 kcal/mol) for wt EIN and Tm = 50 deg C for phosphorylated wt EIN; Tm = 55 deg C ([Delta H] = 130 kcal/mol) for H189A; and Tm = 53 deg C ([Delta H] = 130 kcal/mol) for H189E. Thus, the mutations at His 189 decreased the thermal stability of EIN and the introduction of a negative charge at the active site of EIN was the most destabilizing. Similar studies will be conducted on the same active-site mutants of intact enzyme I. In addition, isothermal titration calorimetry will be used to measure affinity changes for HPr produced by Ala and Glu substitutions for His at position 189. Ultracentrifugation is being used also to measure hydrodynamic properties of mutant and wild-type proteins.
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