Abstract
During translocation across the cytoplasmic membrane of Escherichia coli, glucose is phosphorylated by phospho-IIA(Glc) and Enzyme IICB(Glc), the last two proteins in the phosphotransfer sequence of the phosphoenolpyruvate:glucose phosphotransferase system. Transient state (rapid quench) methods were used to determine the second order rate constants that describe the phosphotransfer reactions (phospho-IIA(Glc) to IICB(Glc) to Glc) and also the second order rate constants for the transfer from phospho-IIA(Glc) to molecularly cloned IIB(Glc), the soluble, cytoplasmic domain of IICB(Glc). The rate constants for the forward and reverse phosphotransfer reactions between IIA(Glc) and IICB(Glc) were 3.9 x 10(6) and 0.31 x 10(6) m(-1) s(-1), respectively, and the rate constant for the physiologically irreversible reaction between [P]IICB(Glc) and Glc was 3.2 x 10(6) m(-1) s(-1). From the rate constants, the equilibrium constants for the transfer of the phospho-group from His90 of [P]IIA(Glc) to the phosphorylation site Cys of IIB(Glc) or IICB(Glc) were found to be 3.5 and 12, respectively. These equilibrium constants signify that the thiophospho-group in these proteins has a high phosphotransfer potential, similar to that of the phosphohistidinyl phosphotransferase system proteins. In these studies, preparations of IICB(Glc) were invariably found to contain endogenous, firmly bound Glc (estimated K'(D) approximately 10(-7) m). The bound Glc was kinetically competent and was rapidly phosphorylated, indicating that IICB(Glc) has a random order, Bi Bi, substituted enzyme mechanism. The equilibrium constant for the binding of Glc was deduced from differences in the statistical goodness of fit of the phosphotransfer data to the kinetic model.
Highlights
The bacterial phosphoenolpyruvate:glycose phosphotransferase system (PTS)3 comprises dozens of cytoplasmic and membrane proteins, most of which are the sugar-specific components of the system
During translocation across the cytoplasmic membrane of Escherichia coli, glucose is phosphorylated by phospho-IIAGlc and Enzyme IICBGlc, the last two proteins in the phosphotransfer sequence of the phosphoenolpyruvate:glucose phosphotransferase system
The data from the phosphotransfer reactions from [P]IIAGlc to Glc via IICBGlc were fitted to Reactions IV, V, VI, and VII
Summary
The bacterial phosphoenolpyruvate:glycose phosphotransferase system (PTS) comprises dozens of cytoplasmic and membrane proteins, most of which are the sugar-specific components of the system (for reviews, see Refs. 1– 4). We have obtained, for the first time, the apparent equilibrium constants for the phosphotransfer reactions between the His residue in IIAGlc and the Cys residue in domain IIB, from which the phosphotransfer potential of a phospho-SCys protein can be derived. Whereas the phospho-S-Cys structure is relatively rare, it is found in the protein-tyrosine phosphatases of eukaryotic cells [8]
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