the involvement of the arginine 17 residue in the active site of the

THE JOURNAL OF BIOLOGICAL CHEMISTRY 0 1993 by The American Society for Biochemistry and Molecular Biology, Inc.

Vol. 268, No. 17, Issue of June 15, pp. 12325-12333.1993 Printed in U. S. A.

The Involvement of the Arginine 17 Residue in the Active Site of the Histidine-containing Protein, HPr, of the Phosphoeno1pyruvate:Sugar Phosphotransferase System of Escherichia coZi*

(Received for publication, December 3, 1992)

J. William Anderson, Katherine Pullen+, Fawzy Georgesg, Rachel E. KlevitST, and E. Bruce Waygoodl1 From the Department of Biochemistry, University of Saskatchewan, Saskatoon, Saskatchewan S7N 0 WO, Canada, the $.Department of Biochemistry, University of Washington, Seattle, Washington 98195, and the W a n t Biotechnology Institute, National Research Council, Saskatoon, Saskatchewan S7N OWO, Canada

Histidine-containing protein, HPr, of the Esche- richia coli phosphoeno1pyruvate:sugar phosphotransferase system has an active site His-15 that is phosphorylated to form M’-P-histidine. The nearby conserved residue, Arg-17, has been replaced by: lysine, histidine, glutamate, glycine, serine, and cysteine. All mutations resulted in impairment of the phosphoaccep- tor function of HPr with enzyme I: kcat/K, values between 6% (Ser-17) and 0.1% (Glu-17), relative to wild type. Several sugar-specific enzymes I1 had different responses. Both the V,, and K , of enzyme

only K , was affected, except for R17E. For both enzymes, kc.JKm values were between 0.5 and 3%, with R17E being 10-fold lower. Except for R17E, minimal effects were observed for enzyme IImannito’ . These results suggest that there are different rate-limiting steps in the enzymes 11. Phosphohydrolysis properties and the pKa values for His-15 and phosphorylated His-15 determined by NMR for both wild type and mutant HPrs suggest that Arg- 17 is partly responsible for the instability of P-His-15 and the depressed pK, values in wild type HPr. Other feature($) of the tertiary structure influence the protonation of His-15 and the phosphohydrolysis properties of phosphorylated His- 15.

IIN-acetylglucoaamine were altered, while for enzyme 11”””””

The histidine-containing protein, HPr,’ is a phosphocarrier protein of the phosphoeno1pyruvate:sugar phosphotransferase system (PTS). It is the acceptor of a phosphoryl group from enzyme I, and the donor of the group to a sugar-specific IIA domain that may exist as a separate phosphocarrier protein or as part of the membrane-bound enzyme I1 that carries out both sugar phosphorylation and translocation (see review by

* This work was supported in part by operating grants from the Medical Research Council of Canada (to E. B. W.) and by National Institutes of Health Grant DK-35187 (to R. E. K.). The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked “aduer- tisement” in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

ll Supported by an American Heart Association Established Inves- tigator Award.

11 To whom correspondence and reprint requests should be ad- dressed. Tel.: 1-306-966-4381; Fax: 1-306-966-8718; E-mail: way- [email protected].

The abbreviations used are: HPr, histidine-containing phosphocarrier protein of the PTS; P-HPr, phospho-HPr; PTS, pbosphoen- o1pyruvate:sugar phospbotransferase system; EI, enzyme I of the PTS; glc, glucose; man, mannose; mtl, mannitol; nag, N-acetylglucosamine; MES, 2-(N-morpholino)ethanesulfonic acid.

Meadow et al. (1990)). The nomenclature proposed by Saier and Reizer (1992) is used in this paper. IIABUgaT domain in its separated form was previously called factor IIIsuga’. There are three-domain membrane-bound enzymes, IIABC (e.g. enzyme IIN-scetylglueosamine ) and two-domain enzymes, IIBC (e.g. enzyme IIglueose ). HPr is a substrate of enzyme I, while phospho-HPr (P-HPr) is the substrate of the various IIA domains, and, in both cases, normal Michaelis-Menten kinetics can be dem- onstrated (Waygood, 1987). The early work of Anderson et al. (1971) showed that the phosphoryl group in P-HPr is carried in the form N*l-P-histidine, whose properties were more fully described by Waygood et al. (1985). This led to the proposal that the active site of Escherichia coli HPr had an arrangement that involved residues His-15, Arg-17, and Glu-85 (Way- good et al., 1985, 1989). Implicit in the term “active site” is the recognition that although HPr can be kinetically charac- terized as a substrate, it probably does participate in a catalytic manner to facilitate the phosphoryl transfer. The initial descriptions of the tertiary structure of E. coli HPr by both two-dimensional NMR (Klevit and Waygood, 1986) and x- ray diffraction (El-Kabbani et al., 1987) showed that the glutamate residue closest to the active site was the C-terminal residue, Glu-85. Replacement and deletion of Glu-85 by site- directed mutagenesis suggests that the C-terminal a-carboxyl may serve a minor role in the phosphoryl transfer mechanism, while the side chain y-carboxyl appears unimportant (Ander- son et al., 1991). The structures of HPrs from Bacillus subtilis and Streptococcus faecalk (Wittekind et al., 1990, 1992; Herz- berg et al., 1992; Jia et al., 1993) have the C-terminal a- carboxyl away from the active site.

Arg-17 is located in the first a-helix in all HPrs and is usually located on the other side of the a-helix from His-15 which N-caps the a-helix. Arg-17 is conserved in all HPrs that have been sequenced (Gonzyl-Treboul et al., 1989; Meadow et al., 1990; Eisermann et al., 1991; Titgemeyer, 1991), and it is reported that its chemical modification with cyclohexanedione yields an HPr with about 20% activity (Kalbitzer et al., 1982). In the x-ray structure of HPr from B. subtilis, the Arg-17 guanido group interacts with a sulfate anion that is close to the His-15 imidazole ring. It has been suggested that this arrangement is an analogue of the phosphorylated form of HPr (Herzberg et al., 1992). The interaction between arginyl side chains and phosphoryl groups in phosphoryl transfer mechanisms has been discussed by Knowles (1980), and such a role was proposed for Arg-17 in HPr by Waygood et al. (1985). In addition to the putative importance of Arg-17 in the phosphoryl transfer mechanism, it has also been proposed that the Arg-17 residue is necessary

12325

12326 Active Site of HPr

for both the unusual pK,, values (Kalbitzer et ai., 1982) exhib- ited by His-15 in E. coli HPr (pK, 5.6-5.7) and P-HPr (pK, 7.7-7.8) (Dooijewaard et al., 1979; Kalbitzer et al., 1982; An- derson et al., 1991) and for the phosphohydrolysis properties of P-HPr (Waygood et al., 1985).

In order to evaluate the role of Arg-17 in the phosphoac- ceptor and phosphodonor roles of HPr, in phosphohydrolysis, and in its contribution to the pKa of HPr, a number of amino acid replacements of Arg-17 have been generated and studied. The properties of these mutant HPrs are presented in this paper.

EXPERIMENTAL PROCEDURES

Materials-Restriction enzymes, Klenow fragment, T4 DNA ligase, and 2',3'-dideoxynucleotides were from Pharmacia LKB Biotechnol- ogy Inc. and New England Biolabs. Radiolabeled compounds were purchased from Du Pont-New England Nuclear or Amersham. En- zyme I was purified as previously described by the method of Waygood and Steeves (1980) from E. coli strain WA2127 containing plasmid pTSHIC9, which contained the genes ptsHIcrr and which was gen- erously provided by Dr. B. Erni. Homogeneous E. coli phosphoenolpyruvate carboxykinase was a gift from Dr. H. Goldie. V8 protease (endoproteinase C) was a product of Miles Scientific. Dithiothreitol, Dowex resins, and Bio-Gel P2 were from Bio-Rad. Urea was electro- phoresis grade from Fisher Biotech. Imidazole and DzO were from Sigma; NaOD and DCl were from Aldrich; deuterated sodium 3- trimethylsilylpropionate-2,2,3,3-d~ was from MSD Isotopes.

Bacterial Strains and Growth-Salmonella typhimwrium strain SB2950 trpB233A(trzAptsHIcrr)49 and strain SB3507 trpB233 were used as sources of enzyme 11""" and enzyme IF"*', respectively (Way- good et al., 1979; Stock et al., 1982). E. coli strain ZSClO3 glk7rpsLptsG2 and strain ZSC114L glk7rpsLptsG2ptsMI were used as sources of enzyme 11""" and enzyme IInaE, respectively (Curtis and Epstein, 1975; Stock et al., 1982). We are grateful to Dr. Epstein for the ZSC strains. E. coli strain CSH4 trprpsL was used as a source of enzymes 11"" and IInS (Anderson et al., 1991). The strains were grown on minimal salts A media (Waygood et al., 1979), with either 0.2% N-acetylglucosamine, mannose, or mannitol, and harvested in midlog phase, except for S. typhimuriurn strain SB2950, which was grown on minimal salts A medium with 0.4% DL-lactate and harvested in stationary phase. Tryptophan (20 pg/ml) was added where required.

Enzyme Assays-The assays for enzyme I, enzyme II""", enzyme I F , and enzyme IImt' were carried out as described previously (An- derson et al., 1991).

Preparation of Phosphorylated Peptides-Preparations of [32P]HPr and [32P]HPr(R17G) at approximately 1 mg/ml were incubated with V8 protease (0.1 mg/ml) in 1 ml of 0.1 M Bicine buffer, pH 8.6, with 1 mM EDTA for 1.5 h at 37 "C. During this incubation, approximately 50% of the phosphoryl group was lost due to hydrolysis. The wild type phosphopeptide preparation was separated from the free phosphate by passage through a 1.5-ml Dowex resin column (AG 1-X8, 50-100 mesh, chloride form) and eluted with water. The R17G phosphopeptide was separated from Pi by passage through a 1 X 20 cm Bio-Gel P2 (200-400 mesh) column equilibrated with 2 mM sodium carbonate and 20 mM KC1 at 4 "2.

Phosphohydrolysis-The phosphorylation and phosphohydrolysis of the HPrs was carried out as described by Anderson et al. (1991). Phosphohydrolysis was carried out on peptides generated by V8 protease digestion of P-HPr and P-HPr(R17G). In order to separate P-peptide from phosphate, the DEAE-cellulose paper chromatography conditions (Waygood et al., 1985) were changed. For wild type HPr (R17) peptide, the chromatography conditions were: replacement of DEAE-cellulose paper with phosphocellulose paper (Whatman P81); chromatography buffer 60% ethanol, 40% water, 10 mM MES, pH 6.4 at 4 "C. For R17G HPr peptides the chromatography conditions were: DEAE-cellulose paper; chromatography buffer, 50% ethanol, 50% water, 10 mM Tris-HC1, pH 7.5, 20 mM KC1 at room temperature.

Determination of N*'-P-histidine and N2-P-histidine by Alkaline Hydroly~is-[~*P]HPr and [32P]enzyme I were produced as described previously (Waygood, 1986; Anderson et al., 1991). These preparations were hydrolyzed at 100 "C in 3 N NaOH for 4 to 24 h to ensure complete hydrolysis. The hydrolysates were diluted 20-fold and applied to a 1.5 X 10 cm Dowex resin (AG 1-X8, 50-100 mesh, bicar- bonate form) column, and eluted (1 ml/min) with a 1L gradient, 0.01

M to 1 M KHCO, as previously described by Anderson et nl. (1971) and Weigel et al. (1982).

HPr Cloning and Site-directed Mutagenesis-The HPr gene was isolated and manipulated as previously described (Anderson et al., 1991). The site-directed mutagenesis was carried out by the general methods of Zoller and Smith (1984) with the modification described by Kunkel (1985). The primers were produced using an Applied Biosystems 380A DNA synthesizer using standard phosphoramidate chemistry (Mateucei and Caruthers, 1981). Sequencing was carried out according to the method of Sanger et al. (1977), and the complete gene for HPr was always sequenced. For protein production, the HPr gene was transferred from MlBmpll to pUC13 using restriction enzyme digestions of Hind111 and EcoRI. The primers used were as fOllOwS: R17C, 5"GGCAGCAGGGCAGGTGTG-3'; R17E, 5'-GGC- AGCAGGCTCGGTGTGCAG-3'; R17G, 5"GGCAGCAGGCCCGG- TGTGCAG-3'; R17H, 5'-GGCAGCAGGGTGGGTGTG-3'; R17K, 5"GGCAGCAGGTTTGGTGTGCAG-3'; R17S, 5"AGCAGGGCT- GGTGTG-3'.

Purification of HPr Proteins-E. coli strain TP2811 F,xylargH11acX74aroBlev*AA(ptsHptsZcrr),KmR, (Levy et al., 1990) transformed by pUC(HPr) was grown as described by Anderson et al. (1991). Purification of HPr mutants from these cells was as described by Anderson et al. (1991). This procedure produced 100-300 mg of homogeneously pure HPr as judged by isoelectrofocusing gels.

Determination of Histidine pK, Values in HPr and P-HPr-The pK, values for the histidine residues of the wild type and mutant forms were determined by measuring the chemical shift of the C2 proton as a function of pH using 'H NMR. All samples contained 50 mM potassium phosphate buffer, 0.2 mM EDTA, 10 p M deuterated sodium 3-trimethylsilylpropionate-2,2,3,3-d~, and 0.5-1.0 mM HPr in DzO. Spectra were collected at 30 "C over 10,000 Hz with presatura- tion of the HDO signal using a 500-MHz AM-Bruker spectrometer. The pD was measured with a combination glass electrode (model MI- 412 from Microelectrodes Inc.) both before and after each spectrum was collected. The electrode was calibrated against standard buffers in HzO. For each sample, 8-14 points were collected over a pH range of approximately 4 to 9, and the pH was changed at each point by the adddition of DCI or NaOD. The chemical shifts were referenced to deuterated sodium 3-trimethylsilylpropionate-2,2,3,3-d~ and fit to the Henderson-Hasselbach equation using a nonlinear least squares analysis. The pK. values obtained were converted to values in Hz0 by subtracting 0.1 f 0.05 pH unit (see below, Determination of the Effects of D20 and Urea on pK. Values). The pK, values for each of the wild type and mutant forms of P-HPr were similarly determined, except that the samples also contained 5 mM MgCl,, approximately 15-30 mM phosphoenolpyruvate, 0.2 mM dithiothreitol, and 0.5 mg/ml enzyme I to phosphorylate the protein at His-15. In all cases, incu- bating at 37 "C for 2 min was sufficient to drive the phosphorylation reaction to completion, and the phosphorylated form was maintained throughout the course of the titration. For these samples, 17-21 points were collected over a pH range of 4 to 10.

Determination of Histidine pK, Values in Unfolded HPr-Deuter- ated urea was prepared by dissolving urea in DzO, heating to 40 "C for 2 h, lyophilizing, and resuspending in DzO to a concentration of 6 M. HPr (1 mM, 0.6 ml) in the same buffer described above was lyophilized and resuspended in 0.6 ml of 6 M urea. The sample was incubated at 40 "C for 1 h, and the pK, was determined by NMR spectroscopy as described above. The values obtained were corrected for the effects of urea by subtracting 0.23 f 0.06 pH unit and for the effects of DzO by subtracting an additional 0.10 f 0.05 pH unit (see below).

Determination of the Effects of D20 and Urea on pKa Values-The pK, of the C2 proton of imidazole in DzO, HZO, and DzO containing 6 M urea was determined exactly as described above for HPr samples. The difference in pK. between the DzO and the Hz0 sample was 0.10 f 0.05 pH unit, and the difference in pK. between the DzO and the D,O with 6 M urea was 0.23 f 0.06 pH unit.

Protein Determinations-HPr concentration was determined by the lactate dehydrogenase depletion assay (Waygood et al., 1979) and the spectrophotometric method of Waddell (1956).

RESULTS

General Properties of Mutant Proteins-To investigate the role that Arg-17 contributes to the activity of HPr, the following site-directed mutants were made: substitution with other basic residues (histidine and lysine, R17H and R17K); an

Active Site of HPr 12327

acidic residue (glutamate, R17E); and smaller, neutral residues (cysteine, glycine, and serine, R17C, R17G, and R17S). All mutant proteins were purified to homogeneity in good yield. No obvious differences were found for any of the mutants with respect to stability or solubility, and the R17C mutant did not dimerize. None of the mutants showed any changes in the binding of the three monoclonal antibodies, Je142, Je144, and Je1323, which indicates that no general structural changes occurred as a result of the substitution of Arg-17 (Sharma et al., 1991; Sharma, 1992).

Kinetics of Enzyme Z and Enzymes ZZ-HPr is a phosphoac- ceptor substrate for enzyme I, and P-HPr is a phosphodonor substrate for various enzymes 11. The enzyme I reaction is: HPr + phosphoenolpyruvate c, P-HPr + pyruvate. The enzymes I1 reactions are: P-HPr + sugar - HPr + sugar-P. Conditions and the reproducibility of the assays of these enzymes have been discussed (Anderson et al., 1991). Enzyme I assays were performed spectrophotometrically by coupling the production of pyruvate to lactate dehydrogenase and NADH. Enzyme I1 assays were performed using excess enzyme I and saturating phosphoenolpyruvate concentration to act as a P-HPr generating system and measuring the formation of [14C]sugar-P in the presence of rate-limiting enzyme 11.

The kinetic results for enzyme I are given in Table I. Replacement of the argininyl residue resulted in a 10- to 40- fold increase in K, and up to a 10-fold decrease in V,,,, yielding k,.,/K,,,values that are about 1% of wild type (Table 11). The R17E mutant suffered a greater loss of activity (kcat/ K, = 0.1%). Surprisingly, R17S was considerably more active than R17K. The dual effects on both K, and Vmnx suggest that Arg-17 may be involved both in the binding of HPr to enzyme I and in efficient catalysis of phosphoryl transfer. Kinetic parameters for various sugar-specific enzymes I1 are presented in Tables I and 111. The enzyme I1 preparations were the same as those previously used to characterize mutants of the C-terminal region of HPr (Anderson et al., 1991). Because of the diversity of results obtained, the experiments were repeated for several of the mutant HPrs using mem- branes from other appropriate strains. These results (see Table 111) are in close agreement with the results presented in Tables I and 11, eliminating the possibility that the diversity observed in the kinetic results is a peculiarity of specific strains. As with enzyme I, the R17E mutation has consider-

ably greater effects upon activity than the other mutations. For enzyme IImt', R17E HPr had a 100-fold increase in K,, while the other mutations showed only minor effects on K,. For enzyme IInag, both K,,, and Vmax effects were observed, indicating that transfer of the phosphoryl group from HPr to the IIAnag domain is potentially rate-limiting. For enzyme II""", the effects were primarily on K,,,. Despite the differences between enzyme 1I"'g and enzyme II""", the kcst/Kn values were similar (Table 11). Many of the K,,, values reported in Tables I and I11 represent high concentrations of HPr, which presented a problem in obtaining kinetic data over a range of appropriate concentrations (-0.2 X K , to 5 X Km). In practice, the highest concentration of HPr mutants that could be obtained in the assays was 1000 to 1500 pM, and thus the high K, values are based upon extrapolations from data obtained at concentrations at or below the K,.

Phosphohydrolysis of Mutant HPrs-The N*'-P-histidine in P-HPrs from various bacterial species have unusual phosphohydrolysis properties (Waygood et al., 1985; Waygood et al., 1988; Anderson et al., 1992). The pH-dependent phosphohydrolysis properties of the free amino acid N*'-P-histidine were determined by Hultquist (1968) who showed that the rates were at least 10-fold higher than those obtained for the free amino acid N2-P-histidine (Table IV). On the basis of studies with modified histidines, Hultquist (1968) concluded that the increased rates of phosphohydrolysis and thus increased instability were due to the interaction of the free cy- amino group of the amino acid with the phosphoryl group. Because this amino group is eliminated by peptide bond formation, the fact that the N*'-P-histidine in P-HPr had phosphohydrolysis rates in excess of the free N*'-P-histidine has been attributed to the interaction between Arg-17 guanido group and the phosphoryl group (Waygood et al., 1985). pH- dependent phosphohydrolysis was carried out on all the Arg- 17 mutants, and, apart from R17H (Fig. lB), all the substi- tutions gave a similar effect, yielding a considerable reduction of rates between pH 2 and 8.5 (Fig. lA). Except for R17H, the "apparent pK,," of this process appears to shift from about 7.7 for wild type to values closer to 8.0 to 8.2, consistent with the more carefully determined pK, values below. Apparent pK, means the pH at which there is a half-maximal rate of hydrolysis. The phosphohydrolysis rates observed for these mutants were similar to those found for the free N*'-P- histidine. The loss of Arg-17 could not be overcome by the

TABLE I Kinetic parameters for HPr mutants

Kinetic parameters are rounded as described by Anderson et al. (1991). Parameters were derived from assays performed at pH 6.8 for enzyme I and pH 7.5 for enzymes 11. Enzyme 11""" was assayed using 1 mM [U-"C]glucose (specific activity of 4,000 cpm/nmol); enzyme 11"" was assayed using 1 mM [U-'4C]-mannitol (specific activity of 2,300 cpm/nmol); enzyme 1I"'was assayed using 1 mM [U-'4C]acetylgluc~~amine (specific activity of 7,200 cpm/nmol).

Enzyme I Enzyme 11"" Enzyme 1I"'g Enzyme 11'"'"

E. coli strain CSH4

E. coli S. typhimurium strain CSH4 strain 2950

K , V,.P K , V'"C.Xb K , V,d P M % P M % P M %

R17, wild type R17K R17H R17G R17S R17C R17E

6 100 15 100 7 100 60 7 30 100 300 60

115 10 35 100 400 30 50 7 30 100 200 50 50 100 300 100

50 30

50 25 30 100 250

300 6 1500 100 2000 9

30

100% activity was 55 pmol of P-HPr produced/min/mg of enzyme I protein. * 100% activity was 1.4 pmol of glucose-6-P produced/min/mg of membrane protein. e 100% activity was 2.7 pmol of mannitol-I-P produced/min/mg of membrane protein.

100% activity was 0.48 rmol of N-acetylglucosamine-6-P produced/min/mg of membrane protein.

P M % 7 100

250 100 1000 100 1000 100 400 100

1500 100 4000 30


addition of arginine, creatinine, NH4Cl, or guanidine HCl (all at 1 M); none of these had an effect on P-Rl7G phosphohydrolysis (pH 6.9, 37 "C).

The results above suggest that Arg-17 has a primary role in causing the increased rates of hydrolysis between pH 2 and 8 as compared to free N6'-P-histidine. This was confirmed by comparing the phosphohydrolysis rates of P-HPr and P- HPr(R17G) in the presence of urea (Fig. IC). All these results suggest that another structural feature of HPr must be inter- acting with the phosphoryl group to yield phosphohydrolysis rates similar to a free Nbl-P-histidine.

Phosphohydrolysis of HPr-derived Peptides-The above results led to the following questions. 1) Is Hultquist's conclusion about the interaction with the free a-amino group during phosphohydrolysis of N"-P-histidine correct? 2) If it is cor-

TABLE I 1 Relative k,,/K, for HPr and mutant HPrs

Values were calculated from Table I.

Enzyme I E. coli E. coli S. typhimurium Enzyme 11"" Enzyme 11""' Enzyme 11"""

strain CSH4 strain CSH4 strain 2950 % %

R17, wild type 100 100 R17K 0.7 50 R17H 0.5 50 R17G 0.8 50 R17S 6 50 R17C 3 50 R17E 0.1 0.1

100 %

3 0.7 0.7 2 0.5 0.05

100 %

1 0.5 2 2 0.7 0.03

TABLE 111 Kinetic parameters for HPr mutants

Parameters were measured as described in Table I. Enzyme 11'"" Enzyme 11""' Enzyme 11""

strain 3507 strain ZSC114L strain zsclO3

K , V,," K , Vma: K , Vmac

S. typhimurium E. coli E. coli

phi % p M % p h f % R17, wild type 16 100 8 100 6 100 R17K 40 100 300 80 300 100 R17G 40 100 300 75 900 100 R17S 40 100 250 100 350 100 a 100% activity was 0.82 pmol of glucose-6-P produced/min/mg of

100% activity was 3.5 pmol of mannitol-1-P produced/min/mg of

e 100% activity was 0.71 pmol of N-acetylglucosamine-6-P pro-

membrane protein.

membrane protein.

duced/min/mg of membrane protein.

rect, what is the behavior of a N*'-P-histidine in a peptide lacking the tertiary interactions of HPr? To address these questions, phosphohydrolysis studies were carried out on a peptide mixture generated by V8 protease digestion which should yield a phosphorylated peptide, residues 6-25. The conditions used had a 10-fold greater V8 protease/HPr ratio than that known to give complete proteolysis at pH 8.0.' The pH was increased to 8.6 to ameliorate the loss of Nbl-P- histidine during the proteolytic incubation in which there was approximately 40-50% survival of the phosphohistidine. To measure the rates of phosphohydrolysis, the chromatography conditions used to separate phosphopeptide and phosphate were altered as described under "Experimental Procedures." The pH-dependent rates of phosphohydrolysis for both peptide preparations were determined and are shown in Fig. 2. The rates are 5- to 15-fold lower than those obtained for P- HPr and are much closer to those that have been obtained for M2-P-histidine in other PTS proteins, but higher than for rates that have been obtained for phosphoimidazole (Table IV). However, the apparent pK, 8.0 for the N"-P-histidine in the peptides appears to be higher than that observed for other phosphoimidazoles (Table IV). This is the same value observed in the phosphohydrolysis experiments with mutant HPrs and suggests that Arg-17 does not interact with P-His- 15 in the peptide.

Identification of the NbL-P-histidine in the Phosphopep- tides-Because the phosphohydrolysis properties of the phosphopeptides were similar to a W2-P-histidine, the possibility that phosphoryl transfer to produce a W2-P-histidine had occurred was tested. Phosphoryl transfer between the two phosphohistidines and histidine amino acids has been dem- onstrated by Hultquist (1968). Phosphohistidines were char- acterized by alkaline hydrolysis followed by ionic exchange chromatography as described under "Experimental Proce- dures." Wild type [32P]HPr and [32P]enzyme I were hydrolyzed to provide standards for Nbl-P-histidine (Anderson et al., 1971) and W2-P-histidine (Weigel et al., 1982), respectively. Hydrolyzed samples from each peptide preparation yielded elution profiles similar to that obtained for HPr and different from enzyme I (Fig. 3). Co-chromatography at various ratios of the hydrolysates of HPr and enzyme I, and of peptides and enzyme I, confirmed that the elution positions of the two isomers of phosphohistidine are different (Fig. 3) as has previously been shown (Anderson et al., 1971; Hays et

* S. Sharma, P. K. Hammen, J. W. Anderson, A. Leung, F. Georges, W. Hengstenberg, R. E. Klevit, and E. B. Waygood, submitted for publication.

TABLE IV Comparison of phosphohydrolysis rates and pK, values

Phosphoamino acid Source Temperature Maximum k" pK.b Ref.

Phosphoimidazole Chemical 39 0.0015 -7 Jencks and Gilchrist (1965) N*'-P-histidine Chemical 46 0.10 -7 Hultquist (1968) N*'-P-histidine E. coli HPr 37 0.12 -7.8 Waygood et al. (1985)

N*l-P-histidine S. faecalis HPr 37 0.06 -7.0 Waygood et al. (1988) P-P-his t idine B. subtilis HPr 37 0.18 -7.2 Anderson et al. (1992) iV2-P-histidine Chemical 46 <0.01 ND Hultquist (1968)

80 -0.1 ND Hultquist et al. (1966) W*-P-histidine E. coli E1 37 0.003 -7 Waygood (1986) W2-P-histidine E. coli IIAgIC 37 0.006 >8 Anderson et al. (1992) W2-P-histidine B. subtilis E1 37 0.003 -7 Anderson et al. (1992) N2-P-histidine B. subtilis IIAgl' 37 0.010 -8 Anderson et al. (1992)

"C min"

46 0.25 ND'

a The maximum rate a t pH > 4.0.

e ND, not determined. For dependence at pH > 5.0.


0.14

0.12

0.10

0.M

0.08

0.04

0.W

0.m

0.14

0.12

0.10

0.08

0.08

0.04

0.02

0.00

, ~~ ~

1 2 3 4 5 6 7 8 0 1 0 1 1

PH

1 2 3 4 5 6 7 B 0 1 0 1 1

1 2 3 4 5 6 7 8 0 1 0 1 1

PH FIG. 1. pH-dependent phosphohydrolysis of HPrs. Phos-

phohydrolysis of P-HPr and phosphorylated mutant HPrs was carried out at 37 “C, and the rates were determined as described under “Experi- mental Procedures.” A , wild type HPr, R17G, o ” - o ; R17K, 1. B, wild type HPr, ..”.; R17E, M R17H, .“-.. C, after denaturation in urea: wild type HPr, c“., R17G, o“--o. The denaturing conditions were: 5 M urea in 2 mM sodium carbonate and 20 mM KC1 (the solution in which [32P]HPr was isolated) at 0 ‘C for 4 h. All the buffers used for the phosphohydrolysis were made up with 5 M urea.

aL, 1973; Weigel et al., 1982). In these previous reports, the incubations used for alkaline hydrolysis were 3-4 h. At these incubation times, we found that considerable phosphorylated material eluted after the phosphohistidines. This material, presumably a mixture of incompletely hydrolyzed phosphopeptides, could be completely eliminated by extending the hydrolysis to 12-24 h depending upon the samples. The longer incubation times produced more Pi, but at all times (4-24 h) of alkaline hydrolysis, the phosphohistidine identified in a particular protein or peptide remained the same. The results shown (Fig. 3) are for 24-h incubations for all samples.

pK. Values of Histidine Residues-The pK, values for the histidine residues in wild type HPr were determined in the

PH FIG. 2. pH-dependent phosphohydrolysis of phosphopep-

tides. Phosphohydrolysis rates were determined as described under “Experimental Procedures” for the phosphopeptides derived from V8 protease digestion of wild type HPr (c”.) and R17G (U).

native form and under denaturing conditions. The latter measurements were made in order to determine the effects of the tertiary structure on the pK,. Under denaturing conditions, the 2 histidines, residues 15 and 76, had similar pK, values of 6.5 and 6.7, and it is not possible to ascertain to which residue these values correspond. However, in native wild type HPr, the pKa values observed for His-15 and His- 76 (Table V) are significantly lower than either of these values, with His-15 having the larger change in pK,, of at least 1.1 pH unit. It should be noted that the differences in the pK, values reported here for native wild type HPr and in earlier publications (Dooijewaard et al., 1979; Kalbitzer et al., 1982; Anderson et al., 1991) can be accounted for by the correction that has been applied for the effects of DzO (see “Experimental Procedures”).

Similar measurements were made on the Arg-17 mutants of HPr. In all cases, there is little or no effect on the pK, of His-76, while an increase in the pK, of His-15 is observed. When Arg-17 is replaced by a neutral side chain (R17G or R17S), the increase is 0.5 pH unit, while for a partly positively charged residue (R17H) it is 0.3 pH unit, and for a fully positively charged side chain (R17K) it is within 0.1 pH unit of the wild type value. Thus, it appears that the depressed pK, of His-15 in wild type HPr can be partly explained by the presence of a positive charge at residue 17, and for a fully positively charged residue the effect is approximately 0.5 pH unit. Interestingly, the replacement of Arg-17 with a negatively charged side chain (R17E) increased the pK,, only slightly more than the neutral mutants; the change in pK, was 0.6 rather than 0.5 unit.

Similar measurements were made on the phosphorylated wild type and mutant forms of HPr (Table V). Again, little or no change in the pK, was observed for His-76, while an increase of 0.3-0.6 pH unit was observed for His-15 when compared to the wild type. However, in this case, the increase for the R17K mutant was in the same range as for the mutants that introduce neutral and negatively charged side chains at residue 17, which suggests that in P-HPr there is a specific effect of the argininyl residue on the pK,. It is also worth noting that the pK, of His-17 in the R17H mutant increases by 0.5 pH unit upon phosphorylation, which is consistent with the idea that the proximity of a charged residue can shift the pKa by -0.5 pH unit.

DISCUSSION

The mutations of residue Arg-17 that are described in this paper were produced to help elucidate the mechanism of phosphoryl transfer in the PTS. The three-dimensional struc-


FIG. 3. Identification of phosphohistidine in the phosphopeptides. [32P]HPr, [32P]enzyme I, 32P-phosphopeptides from wild type HPr, and 32P- phosphopeptides from R71G HPr were incubated for 24 h at 100 "C with 3 N NaOH. The hydrolysates were then chromatographed as described under "Experimental Procedures," and 10-ml fractions were collected. The samples shown are: HPr, N61-P-histidine standard ( A ) , Enzyme I, M2-P-histidine standard ( B ) , HPr and enzyme I ( C ) , peptide derived from wild type HPr (D), HPr and wild type peptide ( E ) , and En- zyme I and wild type peptide (F) . The large peak that eluted at about fraction 37 was Pi. The hydrolysate of the phosphopeptide from R17G behaved identi- cally with the hydrolysate derived from wild type, and the results are not shown.

m 0 w

X

E P V

2 0 3 0 4 0 5 0 6 0 7 0

2 0 3 0 4 0 5 0 6 0 7 0

4 . , .D. wt pe'ptibe '

2 0 30 4 0 5 0 6 0 7 0

2 0 3 0 4 0 5 0 6 0 7 0

4 - . I . , . I .

. F. w i peptide + Enzyme 'I - 3 -

- 2 -

- 1 -

Z o 30 4 0 6 o 0 2 0 30 4 0 5 0 6 0 7 0

TABLE V HistidinepK. values of wild type and mutant HPrs determined by

NMR HPr P-HPr

His-15 His-76 P-His-15 His-76

R17 (wild type) 5.4 6.0 7.7 6.2 Unfolded" 6.5-6.7 6.5-6.7 NDb ND R17K R17H

5.5 6.0 8.1 6.2 5.7 5.9 (6.0)' 8.0 6.2 (6.5)

R17G 5.9 6.1 8.0 6.2 R17S 5.9 6.1 ND ND R17E 6.0 6.1 8.3 6.2

"HPr was denatured as described under "Experimental Proce- dures."

ND, not determined. ' The values in parentheses are the His-17 pK. values.

ture of HPrs from a number of bacterial species has been determined by either two-dimensional NMR or x-ray diffraction: E. coli (Klevit and Waygood, 1986; El-Kabbani et al., 1987; Hammen et al., 1991; van Nuland et al., 1992; Jia, 1992), B. subtilk (Wittekind et al., 1990, 1992; Herzberg et al., 1992), S. faecalis (Jia et al., 1993), Staphylococcus aureus (Kalbitzer et al., 1991). Recently, structures of E. coli HPr have been determined by x-ray diffraction as a free protein (Jia, 1992) and in a complex with antibody (Prasad et al., 1993). As had been predicted (Sharma et al., 1991), these new structural determinations are in agreement with the similar overall folding of other HPrs. Unfortunately, the active site of HPr in the antibody/HPr complex structure at pH 6 is poorly resolved. The free HPr contains a protonated His-15 and a sulfate anion at the active site and is thus an analogue of P- HPr. The active site sequence of HPr is conserved including His-15 and Arg-17 as invariant residues (Meadow et al., 1990). The active site observed in the structures of different HPrs have some common features, but differ in several details. In

FRACTION

all structures, His-15 N-caps the first a-helix, presumably allowing it to interact with the helix dipole (Hol, 1985). The position of the Arg-17 side chain relative to His-15 varies in the different structures. This variability may be attributed to a number of sources, for example: differences in pH (3.7 to 6.5) or ionic strength used during the structure determinations, or the presence of a sulfate ion in the active site, or represent real differences between closely related structures. Despite these differences, the results obtained for mutants of E. coli HPr are likely to have implications for the PTS in other species.

In addition, there are three-dimensional structures determined by either x-ray or two-dimensional NMR of the IIAglc of E. coli (Worthylake et al., 1991; Pelton et al., 1992) and the IIAglC domain of B. subtilk (Liao et al., 1991; Stone et al., 1992), which share considerable homology and with which P- HPr interacts. Recently, van Dijk et al. (1992) determined the state of protonation of the active site histidine of the HAmt' domain. They have concluded that it is difficult to find a common theme for the details of the active site when com- parisons are made to IIAg1" structures. This conclusion is borne out by the kinetic results in this paper.

The activity measurements reported here confirm that Arg- plays an important role in both the acceptor and donor steps of phosphoryl transfer. The loss of the Arg-17 residue in general reduces the catalytic efficiency by lOO-fold, and replacement with an acidic residue antagonizes the situation resulting in a 1000-fold decrease in efficiency. If enzyme I has a ping-pong bi bi mechanism (Waygood, 1986) and not a half- of-sites mechanism (Misset and Robillard, 1982), then the kinetic measurements a t saturating phosphoenolpyruvate concentration are dependent upon rate constants that describe the interaction of HPr:

kl k3

EI-P + HPr (EI-P)(HPr) + E1 + P-HPr (Eq. 1) kz


Despite this seeming simplicity, the K , is not a simple deri- vation (Cleland, 1963): K H P ~ = k (k2 + k d / k l ( k + k d , where k is the rate constant for (EI)(phosphoenolpyruvate) - E1-P + pyruvate. However, it is unlikely that the 10-fold reduction in Vmnx ( i .e . k3) is solely responsible for the 10-fold reductions in KHPr (Table I). Thus, we conclude that Arg-17 is involved in both catalysis and binding interactions of enzyme I. The kinetic situation with the enzymes I1 is more complicated.

The enzymes I1 of the PTS exist in three general arrange- ments. The first two involve enzymes I1 that have either a triple domain (IIABC), for example enzymes I1 for N-acetylglucosamine and mannitol, or an enzyme I1 with a double domain (IIBC) and a separately expressed IIA domain, for example the glucose-specific PTS. The third type is typified by the mannose system in which the IIAB domains are separated from the IIC domain which is in turn comprised of two nonidentical subunits (Meadow et al., 1990). There is often considerable sequence homology between the first two types, but little if any to the third type of enzyme 11. There appear to be mechanistic differences in that the third type involves a phosphohistidinyl residue in the final phosphoryl- transfer step to sugar (Erni et al., 1989), while the former two have a phosphocysteinyl residue (Pas and Robillard, 1988; Pas et al., 1991). There also appear to be differences in kinetic mechanisms used by these enzymes I1 (Grenier et al., 1986). With these considerations in mind, the choice of enzymes I1 to explore the effects of mutation in HPr was based upon the following. Enzyme IInag, which is a IIABC domain enzyme, has a high degree of sequence homology with enzyme II@ (Saier et al., 1988; Peri and Waygood, 1988) and thus allowed a kinetic assessment of the interaction with a IIAglc domain without the added practical complications of a separated IIA domain (see below). Enzyme IImtl is also a IIABC domain enzyme but with very little sequence homology to enzyme IIna8 (Saier et al., 1988) and is probably the most thoroughly investigated enzyme 11. Enzyme 11""" is the third type of enzyme 11, is relatively easy to measure, and is also being thoroughly investigated (Erni et al., 1989). For the IIABC enzymes, a simple mechanistic analysis yields: P-HPr + IIABC - HPr+II(P)ABC - IIA(P)BC + sugar - IIABC + sugar, where each step has rate constants in either direction. The kinetic measurements reported here at saturating sugar concentrations can assess the effects of P-HPr association with the IIA domain, but are probably insensitive to many effects on the catalytic transfer of the phosphoryl group, At present, little is known about the rate-limiting steps. There- fore, depending on the rate-limiting step and its magnitude, the mutants with 100% V,,, values reported in Tables I and I11 could represent instances where there is a significant alteration in phosphoryl transfer ability of HPr, but the sugar phosphorylation step remains rate-limiting. The results indicate clearly that for enzyme IInnK and enzyme 11""" there is significant impairment in function, and an assessment for only enzyme IImt' would have been misleading. However, these results suggest that there may be significant differences in the details of the kinetic mechanisms by which the enzymes I1 catalyze the phosphorylation of sugars, which is supported by previous kinetic results (Grenier et al., 1986), and the conclusions reached by van Dijk et al. (1992) in studying the active site of the IIA"" domain.

To simplify the functional assessment, the HPr/IIA domain interaction should be investigated, and this is possible with a separately expressed IIA domain such as that for glucose. However, in kinetic terms, this complicates the measurement by introducing into the above equation the rate constants that describe the interaction between P-IIA and IIBC. Reizer

et al. (1992) have described kinetic measurements with the IIAgLC domains of E. coli and B. subtilis and report K , values for HPrs that are 10-fold lower than the values reported here. Unfortunately, in some cases, these measurements appear to have been carried out in assays in which the concentrations of IIAg'" and HPr were of the same order of magnitude, and thus one of the usual requirements of steady state kinetics (i.e. [enzyme] << [substrate]) was not met. The effect, if substantial, would be to yield lower estimates of K,.

The in vitro kinetic measurements are not only a tool to understand mechanisms, but they also help evaluate the physiological consequences of these mutations. In our experience, little information is gained from placing a plasmid that causes HPr overexpression into a ptsH strain (Sharma et al., 1991). We are currently modifying a chromosomal gene replacement method to enable a facile HPr chromosomal gene replacement. There is, however, one physiological characterization of an Arg-17 mutant. It has shown that Klebsiellapneumonia strain KAY2034, which is ptsH, has a R17H substitution (Titgemeyer, 1991). HPr from K . pneumonia is identical with E. coli HPr except for an isoleucine to leucine substitution at residue 63 (Titgemeyer, 1991). In K . pneumonia, the pheno- type of the R17H substitution was fermentation-negative on glucitol, reduced fermentation on mannitol, N-acetylglucosamine, and sucrose, and normal fermentation on glucose and fructose. In a comparative assay, based on E. coli enzyme I P l , K . pneumonia R17H HPr had 22% activity, which is similar to the results in Table I. These results along with the changes in kinetic parameters reported here and the large numbers of mutations in HPr that apparently do not affect activity (Sharma et al., 1991; Anderson et al., 1991; Sharma, 1992)' begin to explain why it is difficult to obtain tight HPr mutants. It would appear that the catalytic capacity of HPr and the enzymes with which HPr interacts is far in excess of the physiological demands that are routinely tested.

The above discussion concerns the measurable properties of HPr when it interacts with other proteins, and thus the interpretations are constrained by the limited knowledge about the other proteins. Other experimental approaches, such as phosphohydrolysis and pK, determinations, characterize the mutant HPrs independently of the interactions with other proteins. The phosphohydrolysis results confirm the importance of Arg-17 in increasing the rate at which the phosphoramidate bond in P-HPr is hydrolyzed. With the exception of R17H, which we are currently investigating in more detail, the results suggest that there is a specific require- ment for arginine in increasing rates above those previously described for a N"-P-histidine (Hultquist, 1968). However, Arg-17 is not responsible in HPr for the difference of rates between a N6'-P-histidine and a W2-P-histidine as had been proposed earlier (Waygood et al., 1985). The behavior of N*'- P-histidine in a peptide is clearly similar with respect to rates of phosphohydrolysis to a W2-P-histidine (Fig. 2 and Table IV), and thus suggests that there are other interactions with the imidazole of His-15 in HPr that depend upon the tertiary structure. Many of these interactions may not be described until a tertiary structure of the P-HPr becomes available.

Several studies of phosphoramidate hydrolysis (Jencks and Gilchrist, 1965; Benkovic and Sampson, 1972) have demon- strated that the phosphohydrolysis and phosphoryl transfer properties of P-imidazole deviate from the expected behavior of phosphoramidates of heterocyclic compounds. The reason given is that charge delocalization in the imidazole ring makes the phosphoramidate bond more stable. In native P-HPr, the phosphohydrolysis rates would appear to be close to those expected for a phosphoramidate of a heterocyclic compound


(Benkovic and Sampson, 1972) and as the tertiary structure of HPr is reduced: Arg-17 mutant; brief 5 M urea incubation; V8 peptide isolation; the phosphohydrolysis rates decrease, becoming closer to those observed for a phosphoimidazole. This would suggest that native P-His-15 has a proton that is localized in order to facilitate phosphoryl transfer that is a consequence of the tertiary structure interactions.

It has been suggested that the depressed pKa of His-15 is due to the proximity of the positively charged Arg-17 side chain (Kalbitzer et al., 1982; Waygood et al., 1985). The determinations of the His-15 pKa values in the Arg-17 mutants indicate that the side chain accounts for about half of the ApK. observed for wild-type (ApK, = 0.5 pH unit). Thus, some other structural feature is responsible for the -0.7 pH unit depression of the pK, (relative to unfolded HPr). A likely candidate is the helix dipole, which is proposed to influence pKa values by about 0.5 to 0.8 unit and which has been shown to affect the ionization potential of the imidazole when histidines are placed at either N-terminals or C-terminals of a- helices (Hol, 1985; Sancho et al., 1992). The pK, values for the phosphorylated form of His-15 in the Arg-17 mutants indicates that the Arg-17 side chain is responsible for a pK, depression of -0.3 pH unit. However, this effect appears not to be a simple charge-charge interaction since the replacement of arginine with lysine does not yield a similar lowering of the pK.. Thus, it would appear to be a specific effect of the argininyl side chain. Arginines are known to interact directly with phosphate groups and be important in phosphoryl transfer mechanisms (Knowles, 1980). If this is indeed the case, it is surprising that the effect on the pKa of phosphorylated His- 15 is so small. All the measurements of pK, or apparent pK, in this paper indicate that the pK, of the imidazole in a N61- P-histidine incorporated into a peptide is about 8.0.

Jia et al. (1993) on the basis of the structural determination of S. faecalis HPr have proposed a mechanism for phosphoryl transfer that is based upon the determination that the Arg- 17 guanido group is 18 A from the His-15 imidazole in native HPr. It is thus too far from His-15 to influence the pK,. Our results that show that mutation of Arg-17 affects the pK, of His-15 in native E. coli HPr suggest that Arg-17 side chain is in the proximity of His-15. In the recent E. coli HPr structure (Jia, 1992), the Arg-17 is in a position that is almost identical with that found in S. faecalis HPr, but the His-15 which is bound to a sulfate is in a position very similar to that seen in the B. subtilis HPr (Herzberg et al., 1992). The recent highly refined two-dimensional NMR structure of native B. subtilis HPr shows a propensity for the Arg-17 side chain to be oriented toward His-15, but its position, which is in solvent, is not well defined (Wittekind et al., 1992). It should be noted that the pKa determined for His-15 in native S. fuecalis HPr is about 6.1 (Kalbitzer et al., 1982), which may indicate that the histidine pKa is modified only by the helix dipole effect in S. faecalis HPr.

In this discussion of structure/function relationships in HPr, in particular for the role of Arg-17, there are more questions raised than appear to have been answered. As pointed out earlier, there are differences in the details of the active sites in HPr structures, some of which may be func- tionally relevant. The work in this paper that defines some of the properties of His-15 has given a better description of the “baseline” properties to enable us to assess the effects of various aspects of the tertiary structure. The kinetic results, while highlighting the effects of Arg-17 replacement, suggest that the system is complicated and requires more dissection.

Acknowledgments-We thank Zongchao Jia, Wilson Quail, and Louis Delbaere for allowing on-going access to the structural infor-

mation on the S. fuecalis and E. coli HPr tertiary structures; Phil Hammen for E. coli HPr two-dimensional NMR structural information; and all for stimulating discussions. Alice Leung is thanked for the purification of the HPr proteins.

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