Substitution of Aspartate and Glutamate for Active Center Histidines in the Escherichia coliPhosphoenolpyruvate:Sugar Phosphotransferase System Maintain Phosphotransfer Potential

Abstract

The active center histidines of the Escherichia coli phosphoenolpyruvate:sugar phosphotransferase system proteins; histidine-containing protein, enzyme I, and enzyme IIA Glc were substituted with a series of amino acids (serine, threonine, tyrosine, cysteine, aspartate, and glutamate) with the potential to undergo phosphorylation. Histidine is a unique amino acid with the potential for phosphorylation at two distinct positions 1 , either the N ␦1 or the N ⑀2 atom of the imidazole ring. There are many examples of proteins, both prokaryotic and eukaryotic, in which a histidine is phosphorylated at one of these positions. It has been estimated that phosphohistidine may account for up to 6% of total protein phosphorylations in eukaryotic cells (1). Protein phosphorylations involving phosphohistidines are much more prominent than other phosphoamino acids in organisms such as Escherichia coli and Salmonella typhimurium (2) that utilize sugars via the phosphoenolpyruvate:sugar phosphotransferase system (PTS). 2 Estimates of the concentrations of the PTS proteins and enzymes (3) suggest that internal concentrations of phosphohistidines might exceed 0.2 mM under certain physiological conditions.The PTS functions in sugar phosphorylation and translocation (see reviews in Refs. 4 -6). The PTS system is also involved in a variety of other related cellular functions, such as the regulation of uptake of non-PTS sugars (see review in Ref. 7), chemotaxis (see review in Ref. 8) and catabolite repression (see reviews in Refs. 5 and 6). The PTS is a linear arrangement of both soluble and membrane-bound phosphocarrier proteins and enzymes, which function through a series of phosphotransfer reactions with the phosphate group originating from phosphoenolpyruvate (PEP) and ultimately leading to phosphorylation of the sugar moiety.The first two reactions of the PTS involve enzyme I and histidine-containing protein (HPr), which are soluble, non-sugar-specific, energy coupling components. Two subsequent phosphotransfer reactions involve a membrane-bound, sugar-specific enzyme II. Enzyme II has three or four common functional domains, ABCD (9). The enzyme IIC domain is a membranetransversing domain involved in translocation, and is in some examples complexed with a similar enzyme IID domain. Except for the enzyme IIC and enzyme IID domains, all other protein and enzyme components of the PTS form phosphorylated residues at their active sites in the course of the phosphoryl transfer reactions. The enzyme IIB domain is the site of sugar phosphorylation. Most enzyme II proteins do not have an enzyme IID domain, and in these the phosphoamino acid formed in the enzyme IIB domain is a phosphocysteine (10). In enzyme II, in which an enzyme IID domain is found, the enzyme IIB domain forms a [N ␦1 -P]histidine. The enzyme IIA domain is phosphorylated to form a [N ⑀2 -P]histidine, at residue 90 (11), HPr has a [N ␦1 -P]histidine, at residue 15 (12, 13), and enzyme I a [N ⑀2 -P]histidine, at residue 189 (14). The reactions of the PTS, involving phosp...

Crystal structure of yeast YER010Cp, a knotable member of the RraA protein family

“…The location of this absolutely conserved histidine coincides in Yer010cp with the loop harboring the conserved Arg122 and Asp123. In the E. coli phosphoenolpyruvate: sugar phosphotransferase system, substitution of the active center histidine for aspartate maintains phosphotransfer potential (Napper et al 2001). It would be tempting to hypothesize that Asp123, conserved in most but not all RraA‐like proteins, is involved in a phosphotransfer reaction on a small compound, but no clear residual density supports a covalent link between the compound and Asp123.…”

Selective Extraction and Characterization of a Histidine-Phosphorylated Peptide Using Immobilized Copper(II) Ion Affinity Chromatography and Matrix-Assisted Laser Desorption/Ionization Time-of-Flight Mass Spectrometry

“…Positive ion reflectron MALDI-TOF MS analysis of the unextracted P-HPr digestion mixture (not shown) confirmed the presence of all peptides hypothesized by a theoretical V8 digestion. The P-His peptide VTITAPNGL(pH)TRPAAQFVKE (6)(7)(8)(9)(10)(11)(12)(13)(14)(15)(16)(17)(18)(19)(20)(21)(22)(23)(24)(25) was detected in both phosphorylated (m/z 2230) and nonphosphorylated (m/z 2150) forms with a relative abundance of ∼1:25, indicating that phosphorylation of the histidine residue is unstable under experi- mental conditions. Likewise, analysis of the Cu(II)-IMAC extract of the P-HPr digest solution contained a single sharp peak at m/z 2150 (Figure 1A), corresponding to the predicted mass of the 6-25 peptide without the histidine phosphate group (HPO 3 ).…”

“…Acetonitrile (HPLC grade) was supplied by EM Science (Gibbstown, NJ), and deionized water was prepared on site using the Milli-Q system from Millipore (Bedford, MA). Enzyme I, enzyme IIA glc , and HPr proteins from E. coli were purified to homogeneity using previously published protocols, [23][24][25] as was the CheY protein from Salmonella typhimurium. 26 Phosphorylation Reactions.…”

Glans resurfacing for precancerous and superficially invasive carcinomas of the glans penis: Pathological specimen handling and reporting

“…Enzyme I, Enzyme IIA glc , and HPr proteins from E. coli were purified to homogeneity using previously published protocols (Anderson et al, 1991;Brokx et al, 2000;Napper et al, 2001), as was the CheY protein from Salmonel la typhi murium (Stock et al ., 1985). Phosp horylati on of HPr was achieved by combining 20 ng of Enzyme I with 10 g (1.1 nmol) of HPr in 20 l volumes of a reaction mixture containing 2 mM MgCl 2 , 5 mM phosphoenolpyruvate, and 20 mM HEPES buffer (pH 7.0).…”