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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 --> Enzyme I --> HPr --> IIAGlc --> IICBGlc --> Glc. (HPr is the first phosphocarrier protein of the PTS.) We previously reported two classes of IIAGlc mutations that substantially decrease the P-transfer rate constants to/from IIAGlc. A mutant of His75 which adjoins the active site (His90), (H75Q), was 0.5% as active as wild-type IIAGlc in the reversible P-transfer to HPr. Two possible explanations were offered for this result: (a) the imidazole ring of His75 is required for charge delocalization and (b) H75Q disrupts the hydrogen bond network: Thr73, His75, phospho-His90. The present studies directly test the H-bond network hypothesis. Thr73 was replaced by Ser, Ala, or Val to eliminate the network. Because the rate constants for phosphotransfer to/from HPr were largely unaffected, we conclude that the H-bond network hypothesis is not correct. In the second class of mutants, proteolytic truncation of seven residues of the IIAGlc N terminus caused a 20-fold reduction in phosphotransfer to membrane-bound IICBGlc from Salmonella typhimurium. Here, we report the phosphotransfer rates between two genetically constructed N-terminal truncations of IIAGlc (Delta7 and Delta16) and the proteins IICBGlc and IIBGlc (the soluble cytoplasmic domain of IICBGlc). The truncations did not significantly affect reversible P-transfer to IIBGlc but substantially decreased the rate constants to IICBGlc in E. coli and S. typhimurium membranes. The results support the hypothesis (Wang, G., Peterkofsky, A., and Clore, G. M. (2000) J. Biol. Chem. 275, 39811-39814) that the N-terminal 18-residue domain "docks" IIAGlc to the lipid bilayer of membranes containing IICBGlc.
Highlights
The phosphoenolpyruvate phosphotransferase system (PTS)2 is a major pathway for the uptake of carbohydrates in the eubacteria [1, 2]
We investigate the effects of two different length truncations (6 or 17 residues) of the N-terminal domain of IIAGlc on phosphotransfer rates with the soluble proteins HPr and IIBGlc, and the membrane proteins IICBGlc from S. typhimurium and E. coli
An earlier study showed that replacement of His75 with Gln in IIAGlc reduced both of the rate constants for the reversible phosphotransfer reaction with HPr by ϳ200-fold [11]
Summary
The phosphoenolpyruvate phosphotransferase system (PTS) is a major pathway for the uptake of carbohydrates in the eubacteria [1, 2]. The PTS 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]. Extensive evidence suggests that [P]IIAGlc is a potent stimulator of adenylate cyclase [3] Both the presence or absence of IIAGlc, and its state of phosphorylation, are of importance for the regulation of cell growth. The Catalytic Role of Amino Acids Close to His90—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 (H75Q) [9], which lies very close to His in the tertiary structure, showed that the mutation reduced the rate constants for phosphotransfer to and from HPr by a factor of 200
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