Abstract
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.
Highlights
The kinetic parameters in vitro of the components of the phosphoenolpyruvate:glycose phosphotransferase system (PTS) in enteric bacteria were collected
A parameter sensitivity analysis will be presented to determine to what extent the results depended on assumptions that were made during the derivation of the rate constants of the elementary steps from the phenomenological kinetic constants measured experimentally
As has been pointed out under “Discussion” of Ref. 11, decreasing RPJTS values with increasing protein concentration suggest increased complex formation between the proteins of the PTS. We investigated this point further by calculating with the kinetic model, using parameter values simulating in vivo and values simulating in vitro at protein concentrations of 2 and 6 mg/ml (Table I), the fraction of the different PTS proteins that was free and the fraction that was complexed with other proteins or boundary metabolites (Table III)
Summary
The kinetic parameters in vitro of the components of the phosphoenolpyruvate:glycose phosphotransferase system (PTS) in enteric bacteria were collected. Kinetic Model of PTS proteins in Salmonella typhimurium (9) and of IICBGlc in E. coli (10) have been modulated in turn to determine the extent to which each of these proteins controls the PTS-mediated uptake rate in vivo These dependences were quantified with so-called flux response coefficients, defined as the percentage change in the uptake rate upon a 1% increase in the enzyme concentration (see “Appendix” for mathematical definitions). A value of greater than unity for this sum is in itself remarkable, since it reflects a higher order than linear dependence of flux on total protein concentration and contrasts strongly with the linear relationship between reaction rate and enzyme concentration usually found in enzyme kinetics Following these experiments, two additional questions are still unresolved. Is it reasonable to assume that this sum increases to values above 1 if flux responses toward these “boundary metabolites” are included?
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