The amino terminal domain of enzyme I (residues 1-258 + Arg; EIN) and full length enzyme I (575 residues; EI) harboring active-site mutations (H189E, expected to have properties of phosphorylated forms, and H189A) have been produced by protein bioengineering. Differential scanning calorimetry (DSC) and temperature-induced changes in ellipticity at 222 nm for monomeric wild-type and mutant EIN proteins indicate two-state unfolding. For EIN proteins in 10 mM K-phosphate (and 100 mM KCl) at pH 7.5, deltaH approximately 140 +/- 10 (160) kcal mol(-1) and deltaCp approximately 2.7 (3.3) kcal K(-1) mol(-1). Transition temperatures (Tm) are 57 (59), 55 (58), and 53 (56) degrees C for wild-type, H189A, and H189E forms of EIN, respectively. The order of conformational stability for dephospho-His189, phospho-His189, and H189 substitutions of EIN at pH 7.5 is: His > Ala > Glu > His-PO3(2-) due to differences in conformational entropy. Although H189E mutants have decreased Tm values for overall unfolding the amino terminal domain, a small segment of structure (3 to 12%) is stabilized (Tm approximately 66-68 degrees C). This possibly arises from an ion pair interaction between the gamma-carboxyl of Glu189 and the epsilon-amino group of Lys69 in the docking region for the histidine-containing phosphocarrier protein HPr. However, the binding of HPr to wild-type and active-site mutants of EIN and EI is temperature-independent (entropically controlled) with about the same affinity constant at pH 7.5: K(A)' = 3 +/- 1 x 10(5) M(-1) for EIN and approximately 1.2 x 10(5) M(-1) for EI. Study holds ProTherm entries: 9566, 9567, 9568, 9569, 9570 Extra Details: amino terminal domain of enzyme I
Submitter: Connie Wang
Submission Date: April 24, 2018, 8:38 p.m.
|Number of data points||10|
|Proteins||Phosphoenolpyruvate-protein phosphotransferase ; Phosphoenolpyruvate-protein phosphotransferase|
|Assays/Quantities/Protocols||Experimental Assay: Tm ; Experimental Assay: dHvH ; Experimental Assay: dHcal ; Experimental Assay: Tm|
|Libraries||Mutations for sequence MISGILASPGIAFGKALLLKEDEIVIDRKKISADQVDQEVERFLSGRAKASAQLETIKTKAGETFGEEKEAIFEGHIMLLEDEELEQEIIALIKDKHMTADAAAHEVIEGQASALEELDDEYLKERAADVRDIGKRLLRNILGLKIIDLSAIQDEVILVAADLTPSETAQLNLKKVLGFITDAGGRTSHTSIMARSLELPAIVGTGSVTSQVKNDDYLILDAVNNQVYVNPTNEVIDKMRAVQEQVASEKAELAKLKDR|