Understanding glucose transport by the bacterial phosphoenolpyruvate:glycose phosphotransferase system on the basis of kinetic measurements in vitro.
The kinetic parameters in vitro of the components of the phosphoenolpyruvate:glycose phosphotransferase system (PTS) in enteric bacteria were collected. To address the issue of whether the behavior in vivo of the PTS can be understood in terms of these enzyme kinetics, a detailed kinetic model was constructed. Each overall phosphotransfer reaction was separated into two elementary reactions, the first entailing association of the phosphoryl donor and acceptor into a complex and the second entailing dissociation of the complex into dephosphorylated donor and phosphorylated acceptor. Literature data on the K(m) values and association constants of PTS proteins for their substrates, as well as equilibrium and rate constants for the overall phosphotransfer reactions, were related to the rate constants of the elementary steps in a set of equations; the rate constants could be calculated by solving these equations simultaneously. No kinetic parameters were fitted. As calculated by the model, the kinetic parameter values in vitro could describe experimental results in vivo when varying each of the PTS protein concentrations individually while keeping the other protein concentrations constant. Using the same kinetic constants, but adjusting the protein concentrations in the model to those present in cell-free extracts, the model could reproduce experiments in vitro analyzing the dependence of the flux on the total PTS protein concentration. For modeling conditions in vivo it was crucial that the PTS protein concentrations be implemented at their high in vivo values. The model suggests a new interpretation of results hitherto not understood; in vivo, the major fraction of the PTS proteins may exist as complexes with other PTS proteins or boundary metabolites, whereas in vitro, the fraction of complexed proteins is much smaller.
Rohwer
J M
JM
Department of Biochemistry, University of Stellenbosch, Private Bag X1, 7602 Matieland, South Africa. jrmaties.sun.ac.za
Meadow
N D
ND
Roseman
S
S
Westerhoff
H V
HV
Postma
P W
PW
D001692:Biological Transport; D002474:Cell-Free System; D003198:Computer Simulation; D004305:Dose-Response Relationship, Drug; D004926:Escherichia coli / Q000201:enzymology; D005947:Glucose / Q000378:metabolism*; D007700:Kinetics; D008956:Models, Chemical; D010731:Phosphoenolpyruvate Sugar Phosphotransferase System / Q000737:chemistry* / Q000378:metabolism; D015398:Signal Transduction
D010731:Phosphoenolpyruvate Sugar Phosphotransferase System; D005947:Glucose