Transient State Kinetics of Enzyme IICBGlc, a Glucose Transporter of the Phosphoenolpyruvate Phosphotransferase System of Escherichia coli

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 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 ϳ10 ؊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.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 (for reviews, see Refs. 1-4). The PTS has several important functions in eubacterial cell physiology in addition to its major role in sugar transport, especially in Gram-positive bacteria (5).When the PTS catalyzes sugar transport, the sugar is phosphorylated as it crosses the membrane, and this process requires 3-6 proteins, depending on the sugar. One example is the Glc transport system in enteric bacteria, shown in Fig. 1.The first two proteins, Enzyme I and HPr, are the so-called general proteins of the system, meaning that they are not sugar-specific. The second pair of proteins, IIA Glc and IICB Glc , are the sugar-specific components. In other cases, the sugar-specific proteins vary from one to four separately encoded proteins. All of the phosphotransfer reactions except the last step (transfer to the sugar) are physiologically reversible. A second point is that the phosphorylation site residues in the PTS proteins are generally His, but in a number of the membrane proteins, the phosphorylation site can be a Cys residue. The phosphoryl group is transferred from PEP through a chain of His proteins to the membrane Enzyme II, where the phosphorylation site can be a His or a Cys (as it is in IICB Glc ), and finally to the sugar. In IICB Glc , the B domain extends into the cytoplasm and contains the phosphorylation site Cys.Our long term goal is to be able to predict the kinetics of Glc uptake by intact cells, and for this purpose it is necessary to obtain the rate constants for each of the reactions in Fig. 1. We established the basic methodology by developing a rapid quench method for determini...

Effects of Mutations and Truncations on the Kinetic Behavior of IIAGlc, a Phosphocarrier and Regulatory Protein of the Phosphoenolpyruvate Phosphotransferase System of Escherichia coli

“…The concentration of the phospho-donor and phospho-acceptor proteins ranged from 30 to 50 nM, similar to those used previously (11,28). could be an anomaly resulting, for instance, from differences in the physical structures of the two membrane preparations, or, could be more significant, reflecting differences in the phospholipid compositions surrounding the Glc transport proteins, or, of the intrinsic properties of the proteins.…”

“…The quench solution for experiments with HPr and IIA Glc was 3 M KOH with 9 M urea, used as one volume of quench to two volumes of reaction mixture (11). For experiments with IIA Glc and IIB Glc the quench solution was 0.3 M KOH, 9 M urea, also used as one volume of quench to two volumes of reaction mixture (28). Analysis of the quenched reactions by gel-filtration chromatography using high-performance liquid chromatography grade columns at pH 12.3 was also as described (11).…”

“…The specificity constant is the ratio: k cat /K m (IIA Glc ) and is identical to the rate constant, k IV , for an enzyme with a ping-pong mechanism (28,30). The data were obtained with sugar phosphorylation assays as described under "Experimental Procedures."…”

“…The data were obtained with sugar phosphorylation assays as described under "Experimental Procedures." IICB Glc in washed membranes was used because it yields more reproducible results than the purified protein (28). …”

Localization of the Substrate-binding Site in the Homodimeric Mannitol Transporter, EIImtl, of Escherichia coli

“…1). The phosphoryl transfer reactions from PEP, via EI, HPr, and the A domain to the B domain of Enzyme II are well understood because three-dimensional structures are available for these proteins combined with detailed pre-steady state kinetic data in the case of the glucose transporter, EII glc , from E. coli (14,15). This type of information is not available for the last phosphorylation step from the B domain to the sugar, bound at the C domain.…”