Understanding Glucose Transport by the Bacterial Phosphoenolpyruvate:Glycose Phosphotransferase System on the Basis of Kinetic Measurements in Vitro

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.In many bacteria, the phosphoenolpyruvate:glycose phosphotransferase system (PTS) 1 is involved in the uptake and concomitant phosphorylation of a variety of carbohydrates (for reviews, see Refs. 1 and 2). The PTS is a group transfer pathway; a phosphoryl group derived from phosphoenolpyruvate (PEP) is transferred sequentially along a series of proteins to the carbohydrate molecule. The sequence of phosphotransfer is from PEP to the general cytoplasmic PTS proteins enzyme I (EI) and HPr and, in the case of glucose, further to the carbohydrate-specific cytoplasmic IIA Glc , membrane-bound IICB Glc (the glucose permease), and glucose. For other carbohydrates, specific enzymes II exist (with the A, B, and C domains present either as a single polypeptide or as multiple proteins, depending on the carbohydrate that is transported), which accept the phosphoryl group from HPr (1-4). Apart from its direct role in the above phosphotransfer and its indirect role in transport, IIA Glc is an important signaling molecule, mediating catabolite repression (reviewed in Refs. 1, 2, and 5). The presence or absence of a PTS substrate affects the IIA Glc phosphorylation state; in the absence of PTS substrate, phosphory...

Control of specific growth rate in Saccharomyces cerevisiae

“…Thus an elasticity of the PTS for glucose of 0.99 can be calculated at an external glucose concentration of 0.2 mM. The elasticity of the PTS system for glucose can also be estimated directly from the detailed kinetic model (Rohwer et al, 2000) as 0.97. These data indicate that in E. coli the glucose transporter controls m at 67 % (or 69 % if an elasticity of 0.97 is used) at the low glucose concentrations obtained in glucose-limited chemostat culture at D50.3 h 21 .…”

“…The elasticity for such a rate equation is equal to the one in equation 18. From the elementary rate constants given in Rohwer et al (2000), it can be calculated that the effect of the internal glucose 6-phosphate concentration is negligible due to the low affinity of the enzyme for this product. Thus an elasticity of the PTS for glucose of 0.99 can be calculated at an external glucose concentration of 0.2 mM.…”

Intramolecular domain–domain association/dissociation and phosphoryl transfer in the mannitol transporter of Escherichia coli are not coupled

“…There have been a number of kinetic studies on the glucose-branch PTS using rapid-quench techniques (29,30). (Note that the kinetics of the intramolecular phosphoryl-transfer reaction studied here is not accessible to classical biochemical methodology.)…”

BDI-modelling of complex intracellular dynamics

“…Mendes, 1997). For some unicellular organisms such as the bacterium Escherichia coli (Rohwer et al, 2000, Wang et al, 2001, Tomita et al, 1999, cf. Ben-Jacob et al, 1997, the yeast S. cerevisiae (Teusink et al, 2000;Rizzi et al, 1997), D. discoideum (Wright and Albe, 1994) and T. brucei (Bakker et al, 1997), and for the red blood cell (Mulquiney and Küchel, 1999) some of the chemical pathways are understood in sufficient kinetic detail to obtain a description of their import and primary processing of glucose.…”