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

Abstract

IIAGlc , a component of the glucose-specific phosphoenolpyruvate:phosphotransferase system (PTS) of Escherichia coli, is important in regulating carbohydrate metabolism. In Glc uptake, the phosphotransfer sequence is: phosphoenolpyruvate 3 Enzyme I 3 HPr 3 IIA Glc The phosphoenolpyruvate phosphotransferase system (PTS) 2 is a major pathway for the uptake of carbohydrates in the eubacteria (1, 2).Sugars that are PTS substrates are phosphorylated as they are translocated across the cell membrane, as illustrated schematically in Fig. 1 for the Glc-specific uptake systems of Escherichia coli and Salmonella typhimurium.The PTS also serves several major regulatory functions, including inhibition of the uptake of several carbohydrates that are not PTS substrates and indirect regulation of carbon metabolism on a wide scale by control of the activity of adenylate cyclase (3). In E. coli, the regulatory function is served largely (1, 2, 4) by the 18.1-kDa phosphocarrier protein of the glucose-specific PTS, IIA Glc (in the older literature, this protein was called III Glc ). Unphosphorylated IIA Glc represses transcription of the uptake systems of three non-PTS carbon sources (lactose, maltose, and melibiose) by inhibiting the uptake of inducer (5), and, in the case of glycerol, which is taken up by facilitated diffusion, by inhibiting glycerol kinase (2). Extensive evidence suggests that [P]IIA Glc is a potent stimulator of adenylate cyclase (3). Therefore, both the presence or absence of IIA Glc , and its state of phosphorylation, are of importance for the regulation of cell growth. The state of phosphorylation of IIA Glc is determined by the relative flux of phospho-groups between it and HPr or IICB Glc , the membrane-associated glucose transporter. Structural studies of IIA Glc by NMR and x-ray crystallography (6 -8) have shown that it comprises two domains: 1) an N-terminal domain that is unstructured in solution with the following amino acid sequence: (Met)-Gly-Leu-Phe-Asp-Lys-Leu-Lys-Ser-Leu-ValSer-Asp-Asp-Lys-Lys-Asp-Thr-Gly; the N-terminal Met is quantitatively removed post-translationally, and is not numbered, and 2) a compact domain that consists of the remaining 150 residues, including the active site, His 90 (9, 10). The present report concerns two aspects of the IIA Glc structure thought to play important roles in the phosphotransfer reactions as follows.The Catalytic Role of Amino Acids Close to His 90 -Transient-state (rapid quench) kinetic methods were adapted to study the phosphotransfer reactions of the PTS (11). Application of these methods to the kinetics of a site-directed mutation of His 75 (H75Q) (9), which lies very close to His 90 in the tertiary structure, showed that the mutation reduced the rate constants for phosphotransfer to and from HPr by a factor of 200.Gln is isosteric with His, and structural studies of the (H75Q) IIA Glc mutant (12) indicated virtually no detectable change around the active site. Two hypotheses were offered to explain the 200-fold decrease in the rate constants. (a) ...

How Phosphotransferase System-Related Protein Phosphorylation Regulates Carbohydrate Metabolism in Bacteria

“…Two possible explanations were proposed: (i) His-75 stabilizes the negative charge of PϳHis-90, or (ii) the active site contains a proton relay network also involving Thr-73. The latter hypothesis was recently falsified by replacing Thr-73 with Ser, Ala, or Val and measuring the effect on the phosphotransfer rates to and from HPr (545). These rates were barely affected by the mutations.…”

Spatial and temporal organization of the E. coli PTS components

“…In fact, the embedding of PTS sugar permeases in the inner membrane made this compartment seem compatible for the association of the putative PTS complex. Reports on the association of the soluble EI and HPr with the cytoplasmic membrane (Ghosh et al, 1989;Ye and Saier, 1995;Dubreuil et al, 1996), as well as the presumed membrane anchor of IIA glc (Wang et al, 2003;Meadow et al, 2006) supported this speculation.…”

NMR of Membrane-Associated Peptides and Proteins

“…Because the amino acid sequence pattern at the N terminus of IIA Glc is reminiscent of the amphipathic pattern observed in exchangeable apolipoproteins, Wang et al [90] demonstrated that the N-terminal region of IIA Glc serves as a membrane anchor. Recent studies confirm that this membrane anchor of IIA Glc is not only essential for phosphotransfer in the glucose pathway [92] but also for inhibiting the activity of other sugar transporters such as lactose permease when glucose is present (Fig. 1) [93].…”