Rate and equilibrium constants for phosphoryltransfer between active site histidines of Escherichia coli HPr and the signal transducing protein IIIGlc.
The bacterial phosphoenolpyruvate:glycose phosphotransferase system (PTS) plays a central role in catabolizing many sugars; regulation is effected by phosphorylation of PTS proteins. In Escherichia coli, the phosphoryltransfer sequence for glucose uptake is: PEP --> Enzyme I(His191) --> HPr(His15) --> IIIGlc(His90) --> IIGlc(Cys421) --> glucose. A rapid quench method has now been developed for determining the rate and equilibrium constants of these reactions. The method was validated by control experiments, and gave the following results for phosphoryltransfer between the following protein pairs. For phospho-HPr/IIIGlc (and HPr/phospho-IIIGlc), k1 = 6.1 x 10(7) M-1 s-1, k-1 = 4.7 x 10(7); for the mutant H75QIIIGlc in place of IIIGlc, k1 = 2.8 x 10(5) M-1 s-1, k-1 = 2.3 x 10(5). The derived Keq values agreed with the Keq obtained without use of the rapid quench apparatus. Keq for both reactions is 1-1.5. The rate of phosphoryltransfer between HPr and wild type IIIGlc is close to a diffusion-controlled process, while the reactions involving the mutant H75QIIIGlc are 200-fold slower. These rate differences are explained by an hypothesis for the mechanism of phosphoryltransfer between HPr and IIIGlc based on the structures of mutant and wild type proteins (see Pelton et al. (Pelton, J. G., Torchia, D. A., Remington, S. J., Murphy, K. P., Meadow, N. D., and Roseman, S. (1996) J. Biol. Chem. 271, 33446-33456)).
Meadow
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Department of Biology and the McCollum-Pratt Institute, The Johns Hopkins University, Baltimore, Maryland 21218, USA.
Roseman
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D000942:Antigens, Bacterial / Q000378:metabolism*; D001426:Bacterial Proteins / Q000378:metabolism*; D029968:Escherichia coli Proteins; D006639:Histidine / Q000378:metabolism*; D016297:Mutagenesis, Site-Directed; D010731:Phosphoenolpyruvate Sugar Phosphotransferase System / Q000378:metabolism*; D010766:Phosphorylation; D015398:Signal Transduction
D000942:Antigens, Bacterial; D001426:Bacterial Proteins; D029968:Escherichia coli Proteins; C032242:crr protein, E coli; D006639:Histidine; D010731:Phosphoenolpyruvate Sugar Phosphotransferase System; C030182:phosphocarrier protein HPr