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J. Biol. Chem., Vol. 276, Issue 45, 41588-41593, November 9, 2001

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Substitution of Aspartate and Glutamate for Active Center Histidines in the Escherichia coli Phosphoenolpyruvate:Sugar Phosphotransferase System Maintain Phosphotransfer Potential^*

Scott Napper^§, Stephen J. Brokx^¶, Elliott Pally^¶, Jason Kindrachuk^¶, Louis T. J. Delbaere^¶, and E. Bruce Waygood^¶

From the ^¶ Department of Biochemistry, Health Sciences Building, University of Saskatchewan, 107 Wiggins Road, Saskatoon, SK S7N 5E5 Canada and the  Department of Biochemistry/Veterinary Infectious Disease Organization, University of Saskatchewan, 120 Veterinary Road, Saskatoon, SK S7N 5E3 Canada

Received for publication, May 8, 2001, and in revised form, August 22, 2001

	ABSTRACT

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

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. The mutants [H189E]enzyme I, [H15D]HPr, and [H90E]enzyme IIA^Glc retained ability for phosphorylation as indicated by [³²P]phosphoenolpyruvate labeling. As the active center histidines of both enzyme I and enzyme IIA^Glc undergo phosphorylation of the N² atom, while HPr is phosphorylated at the N¹ atom, a pattern of successful substitution of glutamates for N² phosphorylations and aspartates for N¹ phosphorylations emerges. Furthermore, phosphotransfer between acyl residues: P-aspartyl to glutamyl and P-glutamyl to aspartyl was demonstrated with these mutant proteins and enzymes.

	INTRODUCTION

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

Histidine is a unique amino acid with the potential for phosphorylation at two distinct positions¹, either the N¹ or the N² 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 membrane-transversing 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 [N¹-P]histidine. The enzyme IIA domain is phosphorylated to form a [N²-P]histidine, at residue 90 (11), HPr has a [N¹-P]histidine, at residue 15 (12, 13), and enzyme I a [N²-P]histidine, at residue 189 (14). The reactions of the PTS, involving phosphotransfer from PEP to sugar-P are presented below in Equations 1-5.

(Eq. 1)

(Eq. 2)

(Eq. 3)

(Eq. 4)

(Eq. 5)

In PTS with enzyme IIC and IID domains, an alternating pattern of phosphoryl transfer between the N² to N¹ to N² to N1 atoms of histidines is established. In PTS with only an enzyme IIC domain, the final step involves phosphocysteine.

Another feature of the PTS is that the organization of the enzyme II^sugar complex varies from organism to organism and sugar-specific system to sugar-specific system. Many of the enzyme II^sugar have all their domains as a single gene product. In this study, enzyme IIA^Glc is used, and it is produced as a separate and soluble gene product. The enzyme IIBC^Glc is incorporated into the membrane as a separate gene product.

In the PTS, transfer between phosphohistidines is well established as is transfer from [N²-P]histidine to cysteine. In addition, enzyme I and acetate kinase transfer phosphoryl groups through an interaction between a P-glutamyl residue in acetate kinase and the active site histidine of enzyme I (15). Interactions between [N²-P]histidines and aspartyl residues have been well characterized in the numerous two component regulatory systems (16). This arrangement was mimicked when the mutant [H15D]HPr was created (17).

The active center His-15 of HPr can be mutated to an aspartate ([H15D]HPr) and retain phosphotransfer abilities from EI to EIIA^Glc, although at reduced efficiency (17). Other substitutions, including cysteine, serine, threonine, tyrosine, and glutamate failed to show any phosphotransfer activity. The ability for aspartate to act as a functional substitution for the His-15 N¹ phosphorylation was rationalized on the basis of the similar geometric positioning of the aspartyl carboxyl oxygen atom with the N¹ atom of histidine. Crystallographic determinations of both the wild type and [H15D]HPrs verified the positioning of the aspartate carboxyl to within an Angstrom of the relative position of the N¹ atom of the His-15 imidazole in the wild type structure (17).

In this investigation we determine the potential for functional substitutions of the active center histidines of enzyme I and enzyme IIA^Glc. We demonstrate a pattern of functional substitution of aspartates for histidines that undergo N¹ phosphorylation and glutamates for histidines that undergo N² phosphorylation.

	EXPERIMENTAL PROCEDURES

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

Mutagenesis

Mutants were prepared by a number of methods as previously described (18, 19) but most recently (17) using the QuickChange site-directed mutagenesis kit (Stratagene) according to the manufacturer's instructions.

Protein Expression and Purification

HPr and Mutant HPrs-- HPr protein was overexpressed in the E. coli strain ESK108, F' trp thi rpsL ptsH465 recA56(18), using the pUC19(ptsH) plasmid under the control of the ptsH promoter (20). Homogeneous protein was produced as described previously (20). Isolation of the phosphorylated forms of both the wild type and [H15D]HPr were performed using anion-exchange chromatography in conjuncture with an Amersham Pharmacia Biotech Gradifrac system. Phosphorylation reaction mixtures containing 0.20 mg of HPr or [H15D]HPr and 5 µg of enzyme I were loaded onto a 1 ml mini Q-Sepharose column. The reaction mixture was loaded with 10 mM HEPES buffer pH 7.5, and the column was eluted at 4 ml/min with a 30-ml gradient to 0.1 M NaCl in the same buffer. Fractions (0.5 ml) were collected, and protein elution was monitored at 214 nm. Approximately one-half of the wild type and a third of the [H15D]HPr was isolated in the phosphorylated form in a 1 ml volume.

Enzyme I and Mutant Forms of Enzyme I-- Enzyme I, wild type and mutant proteins, were overexpressed in E. coli strain ESK238, F ompT hsdS_B (r_BmB⁺) dcm gal (DE3), pLysS Cm^r ptsI (23), from ptsI incorporated into pT7-7 (21) at the NdeI restriction site. Purification to homogeneity was as previously described (22, 23).

Enzyme IIA^Glc and Mutant Enzyme IIA^Glc-- The gene for enzyme IIA^Glc, crr, was isolated by polymerase chain reaction from pTSHIC9 (24) and introduced into pT7-7(21) creating pT7-7.crr (17), using the NdeI and BamHI restriction endonuclease sites. Wild type and mutant enzyme IIA^Glc proteins were expressed in E. coli strain ESK262, F ompT hsdS_B (r_BmB⁺) dcm gal (DE3) pLysS Cm^r,pts HIcrr, Kan^r, containing pT7-7.crr (17) following mid-log phase induction by 0.5 mM isopropylthiogalactoside. Large scale overexpression and preparation of crude extracts was identical to that described for enzyme I (21). Purification of enzyme IIA^Glc and mutants was based on the methods previously described (11). Approximately 75 ml of the crude supernatant was applied to a 300 ml Q-Sepharose anion exchange column (3.0 cm × 25 cm), which had been equilibrated with 10 mM potassium phosphate buffer pH 6.5, 1 mM EDTA, and 10 mM p-aminobenzamidine. The column was then eluted using a 2-liter salt gradient of 0-0.5 M KCl and fractions of 10 ml were collected. Fractions containing EIIA^Glc were determined by SDS polyacrylamide gel electrophoresis (SDS-PAGE). EIIA^Glc and mutants were estimated to be >90% pure judged by inspection of SDS-PAGE gels. Appropriate fractions were then pooled and dialyzed overnight against water. Samples were aliquoted and frozen at 20 °C (25).

Production of [³²P]PEP

[³²P]PEP was made by the incubation of 0.2 mCi, of [³²P]ATP (specific activity 3000 Ci/mmol), 0.1 mg of homogeneous E. coli phosphoenolpyruvate carboxykinase (provided by Dr. Hughes Goldie, University of Saskatchewan), 1 mM ATP, 12.5 mM KF, 5.0 mM MgCl₂, and 1 mM oxaloacetate in 50 mM HEPES buffer (pH 7.5) in a final volume of 250 µl at 37 °C for 10 min. The reaction was initiated with the addition of oxaloacetate (25). Reactions were stopped and stored with freezing at 20 °C. The preparation was used without further purification.

Phosphorylation Conditions

Phosphorylation reactions consisted of 5 mM MgCl₂, 25 µM [³²P]PEP (specific activity 800 mCi/mol), and 50 mM HEPES buffer pH 7.5 in a 20-µl volume. Protein concentrations of 5 µg of enzyme I, 10 µg of HPr, and 20 µg of enzyme IIA^Glc were used in all experiments, except were indicated. Mixtures were incubated at 37 °C for 10 min. The reaction was halted with the addition of 2× SDS loading buffer and electrophoresed through a 12% SDS-PAGE gel. Following the run the gels were covered with commercial food wrap, and exposed to x-ray film for 12 h at 80 °C (2).

Mass Spectrometry

Mass spectrometry was performed using a Perseptive Biosystems Voyager ELITE matrix-assisted laser diode ionization-time of flight spectrometer at the Plant Biotechnology Institute. Samples were run in linear mode.

Sugar Phosphorylation Assays

Sugar phosphorylation assays were performed as described (27). Assays were performed in a total volume of 0.05 ml containing 0.05 M potassium phosphate (pH 7.5), 0.1 mM [U-¹⁴C]glucose (10⁵ cpm/mol), 2.0 mM dithiothreitol, 12.5 KF, 10 mM PEP, 5 mM MgCl₂, 0.1 mg HPr, 0.4-1 unit of enzyme II^Man, from SB2950 membranes, as well as varying amounts of enzyme I. The assays were incubated for 30 min at 37 °C and stopped by the addition of 0.1 ml of ice-cold water. The mixtures were loaded onto 1.5 ml columns containing AG1 × 2 (50-100 mesh, Cl form) anion exchange resin equilibrated with water. The columns were washed twice with 5 ml of 1 M LiCl, into scintillation vials fitted 10 ml Nalgene filmware bags. Six milliliters of liquid scintillation mixture was added, and the bags were sealed, shaken, and counted in a liquid scintillation counter.

Other Methods

Methods have been described for protein determinations (18, 20), SDS-PAGE (2), and enzyme I (18, 20, 27).

	RESULTS

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

[H189E]Enzyme I Can Carry Out Phosphotransfer-- The following substitutions for His-189 in E. coli enzyme I were made: aspartate, glutamate, cysteine, serine, threonine, and tyrosine. Each purified mutant was incubated under the phosphorylation conditions described under "Experimental Procedures" either with or without wild type HPr (Fig. 1). The phosphorylation potential of the [H189C]EI mutant was also examined in the absence of dithiothreitol and -mercaptoethanol. Phosphorylation of the active center cysteine of enzyme IIB^Glc is not detectable in the presence of dithiothreitol or -mercaptoethanol.³ Only wild type and [H189E]EI are capable of autophosphorylation and phosphorylation of HPr.

View larger version (77K): [in this window] [in a new window]   Fig. 1.   Phosphorylation of enzyme I His-189 mutants. Enzyme I and/or enzyme I mutants (5 µg) were incubated under phosphorylation conditions alone and with wild type HPr (10 µg). Lanes 1-2, wild type enzyme I; lanes 3-4, [H189D]enzyme I; lanes 5-6, [H189E]enzyme I.

The catalytic activity of [H189E]EI mutant was assessed using the lactate dehydrogenase coupled spectrophotometric assay (27). The autophosphorylation of EI from phosphoenolpyruvate results in a stoichimetric production of pyruvate, which can be coupled to the lactate dehydrogenase reaction. However, even when up to 1 mg of enzyme I was used in an assay that gave maximal activity for the wild type enzyme, no activity for the mutant was detected, and thus no kinetic parameters could be obtained. In the experience of this laboratory, the lactate dehydrogenase assay has a sensitivity limit for EI mutants that retain ~0.01% of wild type activity.

In the sugar phosphorylation assays serial dilutions were performed to determine the limit of detectable activity for enzyme I and H189E enzyme I. Detectable activity was defined as double background. Under this criteria, wild type enzyme I could be detected to femtomolar concentrations while [H189E]enzyme I to nanomolar concentrations. The H189E mutant of enzyme I therefore has ~0.001% of the phosphotransfer activity of the wild type protein. All the other active site mutants of enzyme I failed to promote sugar phosphorylation.

[H90E]EIIA^Glc Can Act as a Phosphoacceptor-- The following substitutions for His-90 in E. coli EIIA^Glc were made: aspartate, glutamate, cysteine, serine, threonine, and tyrosine. Each mutant EIIA^Glc protein was incubated under phosphorylation conditions with both wild type EI and HPr. Only the wild type and H90E mutant of enzyme IIA^Glc demonstrated phosphorylation (Fig. 2).

View larger version (85K): [in this window] [in a new window]   Fig. 2.   Phosphorylation of enzyme IIAGlc His-90 mutants. Enzyme I (5 µg) and HPr (10 µg) were incubated with enzyme IIAGlc and/or [His-90]enzyme IIAGlc mutants (20 µg) under phosphorylation conditions. In addition to the wild type enzyme and HPr each sample contains: Lane 1, wild type enzyme IIAGlc; lane 2, [H90C]enzyme IIAGlc; lane 3, [H90D]enzyme IIAGlc; lane 4, [H90E]Enzyme IIAGlc; lane 5, [His90S]enzyme IIAGlc; lane 6, [H90T]enzyme IIAGlc, and lane 6, [H90Y]enzyme IIAGlc.

The difference in migration of the wild type and H90E enzyme IIA^Glc proteins is not due to differences in molecular mass. The mass of the wild type enzyme IIA^Glc protein is 18,131 Da and the H90E mutant of enzyme EIIA^Glc is 18,120 Da as determined by mass spectrometry (data not shown). This difference in mass corresponds with that predicted by the mutation. The doublet seen in phosphorylation of wild type enzyme IIA^Glc results from the removal of seven residues from the N terminus of the protein as a consequence of an endopeptidase reaction that sometimes occurs in purification. The modification does not affect the ability of the protein to act as a phosphoacceptor (37). The phosphorylation potential of the H90C EIIA^Glc mutant was also examined in the presence and absence of dithiothreitol and -mercaptoethanol. None of the other mutations of EIIA^Glc demonstrated phosphorylation. It was noted however, that in the reaction mixture containing H90D EIIA^Glc there was reduced levels of phosphorylation of both the enzyme I and HPr proteins, as compared with the other phosphorylation reaction mixtures. This could imply that the [³²P]PEP was being depleted from the reaction mixture. Possibly the H90D mutant of EIIA^Glc is able to undergo phosphorylation to form a highly labile phosphoacyl product. Rapid turnover of phosphorylation would account for the reduced levels of phosphorylation of the enzyme I and HPr proteins. However a lactate dehydrogenase coupled spectrophotometric assay comparing turnover rates of phosphorylation in mixtures containing EI (10 µg), HPr (20 µg), and either wild type, H90T, or H90D EIIA^Glc (0.5 mg) failed to demonstrate increased rates of PEP utilization in the mixture containing [H90D]EIIA^Glc as compared with [H90T]EIIA^Glc. This indicates that the [H90D]EIIA^Glc does not undergo phosphorylation to create an unstable phosphoacyl but possibly forms an abortive complex with the other PTS proteins limiting their phosphorylation.

Phosphotransfer between Mutant Enzyme I and Mutant HPr Proteins-- The potential for phosphotransfer between mutant enzyme I and mutant HPr proteins was investigated. A complete investigation of all combinations of the mutants (aspartate, cysteine, glutamate, serine, threonine, and tyrosine) of both His-189 enzyme I and His-15 HPr was performed. Only the acidic substitutions demonstrated successful phosphotransfer and thus only these results are presented. As previously demonstrated, wild type enzyme I is able to phosphorylate both wild type and [H15D]HPr but not [H15E]HPr. The H189E mutant of enzyme I is also able to phosphorylate both wild type and [H15D]HPr (Fig. 3).

View larger version (92K): [in this window] [in a new window]   Fig. 3.   Phosphotransfer from [H189E]enzyme I to [H15D]HPr. Enzyme I or [H189E] enzyme I mutant (5 µg) incubated under phosphorylation conditions with or without HPr and/or HPr mutants (10 µg). Wild type enzyme I with: lane 1, no HPr; lane 2, wild type HPr; lane 3, [H15D]HPr; lane 4, [H15E]HPr. Lanes 5-8 are identical to lanes 1-4 with the substitution of wild type Enzyme I with the [H189E]enzyme I.

Phosphotransfer between Mutant HPr and Enzyme IIA^Glc Proteins-- The ability of phosphorylated [H15D]HPr to transfer the phosphoryl group to both wild type and [H90E]EIIA^Glc was further investigated. To be certain that phosphotransfer was occurring from phospho-HPr and not enzyme I or a contaminating protein, the phosphorylated forms of both HPr or [H15D]HPr were isolated from a phosphorylated reaction mixture. The procedure for this isolation using anion exchange chromatography techniques has been described (17). The isolated phospho-HPr or phospho-[H15D]HPr was incubated with either wild type or one of the mutant enzyme IIA^Glc proteins (Fig. 4). Both the phosphorylated forms of wild type as well as [H15D]HPr were able to mediate phosphotransfer to both wild type and [H90E]enzyme IIA^Glc.

View larger version (64K): [in this window] [in a new window]   Fig. 4.   Phosphotransfer from phospho-[H15D]HPr to [H90E]Enzyme IIAGlc. 32P-phosphorylated forms of wild type and [H15D]HPr (~2 µg) were isolated by anion exchange chromatography and incubated with enzyme IIAGlc or mutant enzyme IIAGlc (20 µg). A, lane 1, wild type enzyme I, HPr, and EIIAGlc; lanes 2-5 all contain wild type P-HPr with lane 2, wild type enzyme IIAGlc; lane 3, [H90D]enzyme IIAGlc; lane 4, [H90E]enzyme IIAGlc; lane 5, [H90T]enzyme IIAGlc. B, identical to A with phospho-[H15D]HPr replacing wild type HPr in lanes 2-5.

Phosphotransfer through a Phosphoacyl PTS-- Phosphotransfer by the first three proteins of the PTS where the active center histidines have been mutated to an acidic amino acid, glutamate for EI and EIIA^Glc, aspartate for HPr was demonstrated (Fig. 5). Phosphorylation from [H189E]enzyme I to [H15D]HPr to [H90E]EIIA^Glc confirms the potential for substitution of histidines that undergo phosphorylation with either aspartates or glutamates.

View larger version (84K): [in this window] [in a new window]   Fig. 5.   Phosphotransfer between mutant PTS proteins. Combinations of wild type and mutant proteins of enzyme I (5 µg) and HPr (10 µg) were incubated with enzyme IIAGlc (20 µg) under phosphorylation conditions. Lane 1, wild type enzyme I, wild type HPr and wild type Enzyme IIAGlc; lane 2, [H189E]enzyme I, [H15D]HPr, and [H90E]enzyme IIAGlc.

	DISCUSSION

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

Phosphorylation at the N¹ or N² imidazole nitrogens of histidine residues is found in many proteins; ATP-citrate lyase (28), phosphoglycerate mutase (29), isocitrate lyase (30), CheA (31), diphosphoglycerate kinase (32) and nucleoside diphosphate kinase (33) to name some. In most cases, the phosphohistidine functions as a high-energy intermediate mediating the passage of a phosphoryl group from a donor to an acceptor molecule. The bacterial PTS is functionally composed of a series of phosphotransfer reactions; enzyme I, HPr, and enzyme IIA^sugar, representing the sequence of the first three phosphotransfer proteins. Phosphorylation of these proteins occurs on histidine residues involving phosphorylations of both the N² and N¹ atoms; N² for enzyme I and enzyme II^sugar and N¹ for HPr. The pattern of phosphotransfer between these PTS proteins therefore proceeds from N² to N¹ to N², and to N¹ for some EIIB^sugar domains.

His-15 in HPr can be substituted with an aspartate and retain the ability for phosphorylation by enzyme I and phosphotransfer to enzyme IIA^Glc. It was proposed that the success of this mutation was due to comparable geometric positioning of the carboxyl oxygen of aspartate to the N¹ atom of histidine (Fig. 6) (17).

View larger version (22K): [in this window] [in a new window]   Fig. 6.   Active center comparison of the wild type and [H15D]HPr x-ray structures. The active centers of wild type (39) and [H15D]HPr (17) are compared by superimposition of the whole HPr structure.

Glutamate has comparable geometric positioning of the O2 carboxyl oxygen to the N² atom of histidine suggesting that glutamate may be a functional substitution for histidines, which undergo phosphorylation of the N² atom such as in enzyme I (Fig. 7) and enzyme IIA^Glc (Fig. 8).

View larger version (23K): [in this window] [in a new window]   Fig. 7.   Modeling of the H189E mutant of enzyme I. The active center of the 259-residue N-terminal domain of wild type enzyme I x-ray structure (40) superimposed on the model of the [H189E]enzyme I. The mutation was modeled by manual introduction of the substitution followed by 40 rounds of least squares refinement.

View larger version (29K): [in this window] [in a new window]   Fig. 8.   Modeling of the H90E mutant of enzyme IIAGlc. The active center of the wild type enzyme IIAGlc 2.1 Å x-ray structure (26) superimposed on the model of the [H90E]enzyme IIAGlc. The mutation was modeled by manual introduction of the substitution followed by 40 rounds of least squares refinement.

Similarly it has been previously noted that asparagine, but not glutamine, may substitute for histidines that are required to hydrogen bond from the N¹ atom (34). The side chain of the asparagine residue may be superimposed such that the asparagine amide group aligns with the N¹ amide of the imidazole ring.

[H189E]EI proved to be the only substitution able to accept a phosphoryl group from PEP and complete phosphotransfer to HPr. The lactate dehydrogenase coupled assay failed to detect measurable activity, indicating that the residual activity was less than 0.01% of wild type. In a more sensitive sugar phosphorylation assay utilizing membranes from a strain deficient in enzyme I it was possible to demonstrate sugar phosphorylation, which was dependent upon the addition of active enzyme I. The H189E mutant of enzyme I was estimated to have a level of phosphotransfer ability of ~0.001% that of wild type. The characterization of numerous other His-189 mutants of enzyme I, which do not display any activity, verifies that the EI-deficient strain contains no contaminating EI activity as previously demonstrated (23).

Similarly, for the mutations of active center His-90 of enzyme IIA^Glc, only the H90E mutation could be phosphorylated. The diminished ability to detect phosphoproteins in reactions containing H90D enzyme IIA^Glc suggested that perhaps this mutant had a spontaneous autophosphohydrolysis activity which we have described for [H15D]HPr (17). Such autophosphohydrolysis activity is also apparent in the two component system e.g., CheY-P has a phosphoaspartyl with a half-life of minutes in the presence of Mg²⁺ (35). This appears not to be the case for [H90D]EIIA^Glc. Also, [H15D]HPr, in the process of dephosphorylation spontaneously forms a protein modification, an isoimide, involving the aspartyl residue (17). However, the inability to detect turnover through a lactate dehydrogenase-coupled assay suggests that this particular mutant of EIIA^Glc interferes with the ability of EI and HPr to phosphorylation, possibly through the formation of an inactive complex.

In this study, the activities of the phosphotransfer reactions utilizing the phosphoacyl derivatives are poor and concerns about contaminating wild type proteins mediating some of the events are addressed here. The proteins and enzymes were expressed in strains that contained suitable mutations. HPr mutants were overproduced in a ptsH strain in which a terminator codon occurs at residue 71⁴ and in which no HPr activity or protein can be detected (38). HPr preparations could not be phosphorylated without the addition of enzyme I. Enzyme IIA^Glc mutants were purified from a pts-HIcrr strain, phosphorylation of expressed wild type enzyme IIA^Glc was dependent on the addition of both enzyme I and HPr. Similarly enzyme I mutants were expressed in a ptsI strain (23). Phosphorylation of the purified enzymes with [³²P]PEP did not reveal any other phosphoproteins present. This however, does not eliminate the possibility of trace amounts of wild type HPr being present and to catalytically shuffle between enzyme I and enzyme IIA^Glc mutants. For this reason, P-HPrs were isolated from enzyme I incubation mixtures to confirm, in particular, the acyl phosphate transfers. Enzyme IIA^Glc preparations could not be phosphorylated without the addition of both enzyme I and HPr, indicating the enzyme IIA preparations were free of contamination by other PTS proteins. The number of results with mutants showing no phosphotransfer, a result expected from a priori considerations, provides additional controls. Lastly, the preparation of [³²P]PEP was a mixture with [-³²P]ATP. None of the results presented here could be reproduced using just [-³²P]ATP.

The creation of two mutant proteins, which are able to form a phosphoglutamyl residue is significant due to the rarity of these linkages in nature. There are few reported examples of phosphoglutamates in the literature; acetate kinase (15) and prothymosin alpha (36). The infrequent observation of phosphoglutamyl residues may in part be due to the instability of these linkages.

Phosphotransfer from histidine to histidine (such as in the PTS), as well as transfer from phosphohistidines to aspartyl residues as seen in two-component systems (31) is well established. The single example of transfer from phosphoglutamate to histidines is acetate kinase to enzyme I (15). We believe there are no examples described in which phosphotransfer occurs between two acidic residues, either phosphoaspartyl to glutamate or phosphoglutamyl to aspartate. Both such reactions have been demonstrated with P-[H189E]EI able to mediate phosphotransfer to [H15D]HPr, which in turn may phosphorylate [H90E]EIIA^Glc. Interestingly, as PTS phosphotransfer requires transfer from N¹ phospholinkages to N², the mutant, acidic phosphotransfers also required alternating residues.

With [H15D]HPr, phosphorylation catalyzed an intramolecular rearrangement in which the phosphoaspartyl side chain cyclizes with the main-chain atoms to produce an isoimide structure. The possibility of such post-phosphorylation activity in the [H189E]enzyme I and [H90E]enzyme IIA^Glc mutants is being investigated. However the additional length of the glutamate, as opposed to aspartate is expected to severely limit such rearrangements, and we have observed no evidence of cyclization of glutamates.

	FOOTNOTES

^* This work was supported by Medical Research Council of Canada Operating Grants MT10162 and MT6147.The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

^§ To whom correspondence should be addressed. Tel.: 306-966-4379; Fax: 306-966-4390; E-mail: Napper@sask.usask.ca.

Published, JBC Papers in Press, September 10, 2001, DOI 10.1074/jbc.M104139200

¹ There are two systems for labeling of the atoms of the imidazole ring of histidine; N1 and N¹ refer to the same nitrogen as do N3 and N².

³ E. B. Waygood, unpublished result.

⁴ J. Lenegler, personal communication.

	ABBREVIATIONS

The abbreviations used are: PTS, phosphoenolpyruvate:sugar phosphotransferase system; PAGE, polyacrylamide gel electrophoresis; PEP phosphoenolpyruvate, EI, enzyme I; HPr, histidine-containing protein; EIIA^Glc, enzyme IIA^glucose.

	REFERENCES

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

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