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

2006; Elsevier BV; Volume: 281; Issue: 17 Linguagem: Inglês

10.1074/jbc.m507417200

1083-351X

Norman D. Meadow, Regina Savtchenko, S. James Remington, Saul Roseman,

Amino Acid Enzymes and Metabolism

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 (Δ7 and Δ16) 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. 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 (Δ7 and Δ16) 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. The phosphoenolpyruvate phosphotransferase system (PTS) 2The abbreviations used are: PTS, phosphoenolpyruvate:glycose phosphotransferase system; IIAGlc, the phosphocarrier protein component of the glucose-specific PTS in E. coli (in the older literature, this protein was named IIIGlc); IICBGlc, the glucose-specific, integral membrane transporter of the PTS; IICBGlc-6His the glucose-specific transporter modified by the addition of 6 histidinyl residues at the C-terminal end; IIBGlc-6His, the cloned domain of IICBGlc containing the phosphorylation site and 6 His residues at the C-terminal end; for all the preceding proteins, the prefixes, [P]or[32P], denote the [P]phospho-protein or [32P]phospho-protein; PEP, phosphoenolpyruvate; HPr, first phosphocarrier protein of the PTS. is a major pathway for the uptake of carbohydrates in the eubacteria (1Meadow N.D. Fox D.K. Roseman S. Annu. Rev. Biochem. 1990; 59: 497-542Crossref PubMed Scopus (302) Google Scholar, 2Postma P.W. Lengeler J.W. Jacobson G.R. Microbiol. Rev. 1993; 57: 543-594Crossref PubMed Google Scholar). 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 (3Peterkofsky A. Reizer A. Reizer J. Gollop N. Zhu P.P. Amin N. Prog. Nucleic Acids Res. Mol. Biol. 1993; 44: 31-65Crossref PubMed Scopus (46) Google Scholar). In E. coli, the regulatory function is served largely (1Meadow N.D. Fox D.K. Roseman S. Annu. Rev. Biochem. 1990; 59: 497-542Crossref PubMed Scopus (302) Google Scholar, 2Postma P.W. Lengeler J.W. Jacobson G.R. Microbiol. Rev. 1993; 57: 543-594Crossref PubMed Google Scholar, 4Seok Y.-J. Sonde M. Badawi P. Lewis M.S. Briggs M.C. Jaffe H. Peterkofsky A. J. Biol. Chem. 1997; 272: 26511-26521Abstract Full Text Full Text PDF PubMed Scopus (82) Google Scholar) by the 18.1-kDa phosphocarrier protein of the glucose-specific PTS, IIAGlc (in the older literature, this protein was called IIIGlc). Unphosphorylated IIAGlc represses transcription of the uptake systems of three non-PTS carbon sources (lactose, maltose, and melibiose) by inhibiting the uptake of inducer (5Saier M.H. Roseman S. J. Biol. Chem. 1976; 251: 6606-6615Abstract Full Text PDF PubMed Google Scholar), and, in the case of glycerol, which is taken up by facilitated diffusion, by inhibiting glycerol kinase (2Postma P.W. Lengeler J.W. Jacobson G.R. Microbiol. Rev. 1993; 57: 543-594Crossref PubMed Google Scholar). Extensive evidence suggests that [P]IIAGlc is a potent stimulator of adenylate cyclase (3Peterkofsky A. Reizer A. Reizer J. Gollop N. Zhu P.P. Amin N. Prog. Nucleic Acids Res. Mol. Biol. 1993; 44: 31-65Crossref PubMed Scopus (46) Google Scholar). Therefore, both the presence or absence of IIAGlc, and its state of phosphorylation, are of importance for the regulation of cell growth. The state of phosphorylation of IIAGlc is determined by the relative flux of phospho-groups between it and HPr or IICBGlc, the membrane-associated glucose transporter. Structural studies of IIAGlc by NMR and x-ray crystallography (6Worthylake D. Meadow N.D. Roseman S. Liao D.-I. Herzberg O. Remington S.J. Proc. Natl. Acad. Sci. U. S. A. 1991; 88: 10382-10386Crossref PubMed Scopus (88) Google Scholar, 7Feese M.D. Comolli L. Meadow N.D. Roseman S. Remington S.J. Biochemistry. 1997; 36: 16087-16096Crossref PubMed Scopus (35) Google Scholar, 8Pelton J.G. Torchia D.A. Meadow N.D. Wong C.-Y. Roseman S. Proc. Natl. Acad. Sci., U. S. A. 1991; 88: 3479-3483Crossref PubMed Scopus (38) Google Scholar) 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-Val-Ser-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, His90 (9Presper K.A. Wong C.-Y. Liu L. Meadow N.D. Roseman S. Proc. Natl. Acad. Sci., U. S. A. 1989; 86: 4052-4055Crossref PubMed Scopus (50) Google Scholar, 10Dörschug M. Frank R. Kalbitzer H.R. Hengstenberg W. Deutscher J. Eur. J. Biochem. 1984; 144: 113-119Crossref PubMed Scopus (62) Google Scholar). The present report concerns two aspects of the IIAGlc structure thought to play important roles in the phosphotransfer reactions as follows. 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 (11Meadow N.D. Roseman S. J. Biol. Chem. 1996; 271: 33440-33445Abstract Full Text Full Text PDF PubMed Scopus (21) Google Scholar). Application of these methods to the kinetics of a site-directed mutation of His75 (H75Q) (9Presper K.A. Wong C.-Y. Liu L. Meadow N.D. Roseman S. Proc. Natl. Acad. Sci., U. S. A. 1989; 86: 4052-4055Crossref PubMed Scopus (50) Google Scholar), which lies very close to His90 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) IIAGlc mutant (12Pelton J.G. Torchia D.A. Remington S.J. Murphy K.P. Meadow N.D. Roseman S. J. Biol. Chem. 1996; 271: 33446-33456Abstract Full Text Full Text PDF PubMed Scopus (18) Google Scholar) indicated virtually no detectable change around the active site. Two hypotheses were offered to explain the 200-fold decrease in the rate constants. (a) His75 stabilizes the negative charge on P-His90 by virtue of a hydrogen bond and, consequently, delocalizes the negative charge on the intermediate (Fig. 2) (12Pelton J.G. Torchia D.A. Remington S.J. Murphy K.P. Meadow N.D. Roseman S. J. Biol. Chem. 1996; 271: 33446-33456Abstract Full Text Full Text PDF PubMed Scopus (18) Google Scholar). This delocalization of the charge would be substantially decreased in the Gln mutant. (b) The active site contains a possible “proton relay” network consisting of Thr73-His75-P-His90. Whether proton transfer might take place between Thr73 and His75 during the phosphotransfer reaction had not been investigated. In the present studies, the validity of the second hypothesis was tested by eliminating the H-bond network by substituting amino acids (Ser, Ala, or Val) for Thr73 but maintaining His75. The rate constants for phosphotransfer between these mutants and HPr were measured. The Function of the Unstructured N-terminal Domain—Early kinetic studies of IIAGlc by steady-state methods showed that [P]IIAGlc is a Michaelis-Menten substrate of IICBGlc with a definable Vmax and Km (13Meadow N.D. Roseman S. J. Biol. Chem. 1982; 257: 14526-14537Abstract Full Text PDF PubMed Google Scholar, 14Meadow N.D. Kukuruzinska M.A. Roseman S. Martonosi A. Enzymes of Biological Membranes. Plenum Press, New York1985: 523-559Google Scholar). A modified form of IIAGlc was isolated that was much less active kinetically (a few percent of wild type) (13Meadow N.D. Roseman S. J. Biol. Chem. 1982; 257: 14526-14537Abstract Full Text PDF PubMed Google Scholar). This protein was a truncated form of IIAGlc, lacking 7 residues at the N terminus. The truncation is catalyzed by what appears to be a specific membrane protease (15Meadow N.D. Coyle P. Komoryia A. Anfinsen C.B. Roseman S. J. Biol. Chem. 1986; 261: 13504-13509Abstract Full Text PDF PubMed Google Scholar). The proteolysis occurs between Lys7 and Ser8 (13Meadow N.D. Roseman S. J. Biol. Chem. 1982; 257: 14526-14537Abstract Full Text PDF PubMed Google Scholar, 15Meadow N.D. Coyle P. Komoryia A. Anfinsen C.B. Roseman S. J. Biol. Chem. 1986; 261: 13504-13509Abstract Full Text PDF PubMed Google Scholar). A function for the N-terminal domain of IIAGlc is suggested by recent studies of the physical properties of the domain. These suggest that it can form an amphipathic helix that serves as a membrane anchor (16Wang G. Keifer P.A. Peterkofsky A. Protein Sci. 2003; 12: 1087-1096Crossref PubMed Scopus (46) Google Scholar, 17Wang G. Peterkofsky A. Clore G.M. J. Biol. Chem. 2000; 275: 39811-39814Abstract Full Text Full Text PDF PubMed Scopus (44) Google Scholar). In the present work, 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. Materials—All buffer salts and other reagents were of the highest purity commercially available. The pH of all buffers is reported at the temperature and concentration at which they were used. Methyl-α-[U-14C]glucoside was synthesized from [U-14C]glucose (NEC 042, PerkinElmer Life Sciences) by the method of Bollenback (18Bollenback G.N. Methods Carbohydr. Chem. 1963; 2: 326-328Google Scholar), and the products were purified by the method of Austin et al. (19Austin P.W. Hardy F.E. Buchanan J.G. Baddiley J. J. Chem. Soc. 1963; : 5350-5353Crossref Google Scholar). The enzymatic synthesis of [32P]PEP (20Roossien F.F. Brink J. Robillard G.T. Biochim. Biophys. Acta. 1983; 760: 185-187Crossref PubMed Scopus (59) Google Scholar) was performed with modifications as described previously (11Meadow N.D. Roseman S. J. Biol. Chem. 1996; 271: 33440-33445Abstract Full Text Full Text PDF PubMed Scopus (21) Google Scholar). Bacterial Strains, Plasmids, and Growth Media—All strains were grown on Luria-Bertani broth containing the required antibiotics and inducers. E. coli strain BL21(DE3) (F– ompT hsdSB (rB– mB–) gal dcm (DE3)) (Novagen) was used as the host for the pET21a plasmids containing the mutated genes for IIAGlc. Construction of Site-directed Mutants of IIAGlc—A mutagenesis method based on a one-step PCR was used to construct plasmids containing deletions of either the first 8 or the first 17 codons of the gene. These codons include the N-terminal Met, which is not assigned an amino acid sequence number because it is removed post-translationally in vivo. Thus, the gene contains 169 codons, but the mature protein contains 168 amino acids, so that the number designating an amino acid residue is smaller than its codon number by one. In the case of the 8 codon deletion, Ser8 was replaced by Met, so that the encoded protein was identical to the proteolytically truncated protein previously isolated, except that Met now replaced Ser as the first residue. This protein is referred to as (Δ7)IIAGlc. To test the effect of a more extensive truncation of the N-terminal domain, Thr17 was replaced by Met, creating a protein that is 16 amino acids residues shorter than the mature, native protein and lacks virtually the entire N-terminal domain of IIAGlc (18 amino acids). This protein is referred to as (Δ16)IIAGlc. The plasmid pDS35 (21Saffen D.W. Presper K.A. Doering T. Roseman S. J. Biol. Chem. 1987; 262: 16241-16253Abstract Full Text PDF PubMed Google Scholar) containing the crr gene was used as the template. PCR was performed by using two sets of primers. For S8M, the primers were: SS112 (5′-GGCGCCATTTTTCACTGCCAGAATTCTTACTTCTTGATGCG-3′) containing the EcoRI site of restriction and S8M (5′-GTTCGATAAACTGCATATGCTGGTTTCCGAC-3′) containing an NdeI restriction site. For T17M the primers were: SS112 and T17M (5′-GACGACAAGAAGCATATGGGAACTATTGAG-3′) containing the NdeI site. After the PCR reaction, PCR products were restricted with NdeI and EcoRI and then cloned into plasmid pET21a (Novagen). Construction of T73S, T73A, and T73V mutants was performed by using the method based on a two-step PCR as described (22Toptygin D. Savtchenko R.S. Meadow N.D. Brand L. J. Phys. Chem. B. 2001; 105: 2043-2055Crossref Scopus (58) Google Scholar). For T73A the primers were: T73A1 (5′-ATCTTTGAAGCCAACCACGCA-3′) and T73A2 (5′-TGCGTGGTTGGCTTCAAAGAT-3′) containing the T73A mutation. For T73S the primers were: T73S1 (5′-ATCTTTGAATCCAACCACGCA-3′) and T73S2 (5′-TGCGTGGTTGGATTCAAAGAT-3′) containing the T73S mutation. For T73V the primers were: T73V1 (5′-ATCTTTGAAGTCAACCACGCA-3′) and T73V2 (5′-TGCGTGGTTGACTTCAAAGAT-3′) containing the T73V mutation. Purification and Characterization of HPr, [32P]HPr, IIAGlc, [32P]IIAGlc, IIBGlc-6His, and [32P]IIBGlc-6His—Homogeneous IIAGlc (23Pelton J.G. Torchia D.A. Meadow N.D. Wong C.-Y. Roseman S. Biochem. 1991; 30: 10043-10057Crossref PubMed Scopus (60) Google Scholar) and HPr (24Beneski D.A. Nakazawa A. Weigel N. Hartman P.E. Roseman S. J. Biol. Chem. 1982; 257: 14492-14498Abstract Full Text PDF PubMed Google Scholar) were isolated as described. The various site-directed mutants of IIAGlc were purified by the same methods used for the wild-type protein. The T73V mutant of IIAGlc behaved somewhat differently during the purification and required a second passage on the ion exchange column to obtain homogeneous protein. IIAGlc was tested for proteolysis by the use of polyacrylamide gel chromatography (13Meadow N.D. Roseman S. J. Biol. Chem. 1982; 257: 14526-14537Abstract Full Text PDF PubMed Google Scholar). IIBGlc-6His was purified by the method of Buhr et al. (25Buhr A. Flükiger K. Erni B. J. Biol. Chem. 1994; 269: 23437-23443Abstract Full Text PDF PubMed Google Scholar), except that a Superose 12 HR 10/30 column (Amersham Biosciences) was substituted for Sephadex G75. The final preparations were apparently homogeneous as judged by SDS-PAGE. The methods previously described (11Meadow N.D. Roseman S. J. Biol. Chem. 1996; 271: 33440-33445Abstract Full Text Full Text PDF PubMed Scopus (21) Google Scholar) were used to determine the concentrations of HPr, IIAGlc, and IIBGlc-6His. [32P]HPr, [32P]IIAGlc, and [32P]IIBGlc-6His were prepared as described previously, with the same attention to the accuracy of the specific activity of the [32P]PEP (11Meadow N.D. Roseman S. J. Biol. Chem. 1996; 271: 33440-33445Abstract Full Text Full Text PDF PubMed Scopus (21) Google Scholar). Sugar Phosphorylation Assay for PTS Activity and the Determination of Specificity Constants—The PEP-driven sugar phosphorylation assay was performed as reported (26Waygood E.B. Meadow N.D. Methods Enzymol. 1982; 90: 423-431Crossref PubMed Scopus (6) Google Scholar, 27Waygood E.B. Meadow N.D. Roseman S. Anal. Biochem. 1979; 95: 293-304Crossref PubMed Scopus (66) Google Scholar). Membrane suspensions (28Meadow N.D. Savtchenko R.S. Nezami A. Roseman S. J. Biol. Chem. 2005; 280: 41872-41880Abstract Full Text Full Text PDF PubMed Scopus (8) Google Scholar) for these assays were prepared from E. coli strain ZSC112ΔG harboring plasmid pCB30 (encoding wild-type IICBGlc) and S. typhimurium, strain PP1133 (29Bouma C.L. Meadow N.D. Stover E.W. Roseman S. Proc. Natl. Acad. Sci., U. S. A. 1987; 84: 930-934Crossref PubMed Scopus (23) Google Scholar). To determine Vmax and Km(IIAGlc), the assay mixture contained fixed concentrations of the following substances in a volume of 0.1 ml: 50 mm Tris/Cl– buffer, pH 8.0; 10 mm KF; 5 mm MgCl2; 10 mm PEP; 5 mm methyl-α-[U-14C]glucoside (5 Bq/nmol); 8 nm Enzyme I (this is 5 units, where a unit is defined as the quantity of enzyme that produces 1 μmol of sugar phosphate in 30 min at 37 °C); and 6 μm HPr. The assays contained <0.2 unit of IICBGlc; the concentration of E. coli IICBGlc was ∼16 nm and S. typhimurium IICBGlc ∼20 nm determined as described (28Meadow N.D. Savtchenko R.S. Nezami A. Roseman S. J. Biol. Chem. 2005; 280: 41872-41880Abstract Full Text Full Text PDF PubMed Scopus (8) Google Scholar). The concentrations of IIAGlc, either wild type or truncated, were 3, 5, 20, and 60 μm. Vmax and Km (IIAGlc) were calculated by the methods of Eadie-Hofstee and Hanes (30Cornish-Bowden A. Fundamentals of Enzyme Kinetics.Rev. Ed. Portland Press, London1995: 33Google Scholar), and the values were averaged. The specificity constant of IICBGlc for IIAGlc is defined as kcat/Km(IIAGlc) (30Cornish-Bowden A. Fundamentals of Enzyme Kinetics.Rev. Ed. Portland Press, London1995: 33Google Scholar), where kcat is defined as Vmax/[IICBGlc]total. We emphasize that the specificity constant is mathematically equal to the rate constant in a reaction catalyzed by an enzyme with a ping-pong mechanism. Rapid Quench Assays—The present study employed the same rapid quench apparatus described previously, including all the details for its set-up and use, treatment with bovine serum albumin to eliminate adsorption of protein, etc. (11Meadow N.D. Roseman S. J. Biol. Chem. 1996; 271: 33440-33445Abstract Full Text Full Text PDF PubMed Scopus (21) Google Scholar). The quench solution for experiments with HPr and IIAGlc was 3 m KOH with 9 m urea, used as one volume of quench to two volumes of reaction mixture (11Meadow N.D. Roseman S. J. Biol. Chem. 1996; 271: 33440-33445Abstract Full Text Full Text PDF PubMed Scopus (21) Google Scholar). For experiments with IIAGlc and IIBGlc the quench solution was 0.3 m KOH, 9 m urea, also used as one volume of quench to two volumes of reaction mixture (28Meadow N.D. Savtchenko R.S. Nezami A. Roseman S. J. Biol. Chem. 2005; 280: 41872-41880Abstract Full Text Full Text PDF PubMed Scopus (8) Google Scholar). Analysis of the quenched reactions by gel-filtration chromatography using high-performance liquid chromatography grade columns at pH 12.3 was also as described (11Meadow N.D. Roseman S. J. Biol. Chem. 1996; 271: 33440-33445Abstract Full Text Full Text PDF PubMed Scopus (21) Google Scholar). Preparation of the solutions for rapid quench experiments required large dilutions from stock solutions of Enzyme I and HPr, and a change from the frozen state to ambient temperature (∼23 °C), at which all experiments were performed. The solutions were therefore incubated for an hour at ambient temperature before the experiment was started. Methods Used to Model the Experimental Data on the Rate of Phospho-group Transfer—The numerical integration program, Kinsim (31Barshop B.A. Wrenn R.F. Frieden C. Anal. Biochem. 1983; 130: 134-145Crossref PubMed Scopus (669) Google Scholar), as modified by Anderson et al. (32Anderson K.S. Sikorski J.A. Johnson K.A. Biochemistry. 1988; 27: 7395-7406Crossref PubMed Scopus (165) Google Scholar), was used to manually fit mathematical models to the experimental data. When experimental data met the criteria for non-linear least squares fitting of mathematical models, the Fitsim (33Zimmerle C.T. Frieden C. Biochem. J. 1989; 258: 381-387Crossref PubMed Scopus (206) Google Scholar) module of Kinsim was used. The model used for the kinetic analysis of the phosphotransfer reactions is shown as Scheme 1. 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 ∼200-fold (11Meadow N.D. Roseman S. J. Biol. Chem. 1996; 271: 33440-33445Abstract Full Text Full Text PDF PubMed Scopus (21) Google Scholar). His and Gln are approximately isosteric and can make approximately the same hydrogen bonds. That is, each has two locations for H-bonding. Depending on orientation either can be a donor or acceptor, and the H-bonding atoms can be in approximately the same spatial location in each case. It has been experimentally determined that in the H75Q mutant, Gln75 is a hydrogen bond donor to the phospho-group, but the bond from Thr73 is lost (12Pelton J.G. Torchia D.A. Remington S.J. Murphy K.P. Meadow N.D. Roseman S. J. Biol. Chem. 1996; 271: 33446-33456Abstract Full Text Full Text PDF PubMed Scopus (18) Google Scholar), i.e. the hydrogen bond network is lost. This result was the basis of one of two hypotheses to explain the 200-fold decrease in activity of the His75 → Gln mutant. The idea was tested by retaining His75 but destroying the H-bond network; amino acids were substituted for Thr73. If such mutants showed a greatly lowered activity, similar to the original (His75 → Gln), this would lend credibility to the idea of a catalytic proton derived from the H-bond network. If the mutants behaved like wild-type IIAGlc, this would be strong evidence supporting the second hypothesis, i.e. the importance of the imidazole ring of His75. The mutants Thr73 → Ser, Ala, and Val were therefore constructed and tested. The results with each of the mutants are summarized and compared with wild-type IIAGlc in Table 1. A progress curve typical of these experiments is shown in Fig. 3.TABLE 1Rate constants for phosphotransfer between HPr and IIAGlc, wild type, and mutantsIIAGlckIIIk-IIInKeq'm-1 s-1 (±S.E.) × 10-6wtaThe values for kIII and k-III, obtained at pH 7.5, are similar to those obtained previously at pH 6.5 (11), which were 61 × 106 m-1 s-1 and 47 × 106 m-1 s-1, respectively.60 ± 1442 ± 831.4T73SbOne of the experiments with this protein was started in the reverse direction, i.e., with HPr as the acceptor and [32P]phospho-IIAGlc as the donor. The rate constants derived from experiments started from opposite directions were in good agreement, indicating that there are no significant concentrations of intermediate complexes between the two reacting proteins prior to the last step, transfer of the phosphoryl group to the acceptor, and separation of the proteins to yield the products.38 ± 1220 ± 331.9T73A44 ± 516 ± 922.8T73VbOne of the experiments with this protein was started in the reverse direction, i.e., with HPr as the acceptor and [32P]phospho-IIAGlc as the donor. The rate constants derived from experiments started from opposite directions were in good agreement, indicating that there are no significant concentrations of intermediate complexes between the two reacting proteins prior to the last step, transfer of the phosphoryl group to the acceptor, and separation of the proteins to yield the products.21 ± 713 ± 531.7Δ7bOne of the experiments with this protein was started in the reverse direction, i.e., with HPr as the acceptor and [32P]phospho-IIAGlc as the donor. The rate constants derived from experiments started from opposite directions were in good agreement, indicating that there are no significant concentrations of intermediate complexes between the two reacting proteins prior to the last step, transfer of the phosphoryl group to the acceptor, and separation of the proteins to yield the products.35 ± 627 ± 1131.3Δ16bOne of the experiments with this protein was started in the reverse direction, i.e., with HPr as the acceptor and [32P]phospho-IIAGlc as the donor. The rate constants derived from experiments started from opposite directions were in good agreement, indicating that there are no significant concentrations of intermediate complexes between the two reacting proteins prior to the last step, transfer of the phosphoryl group to the acceptor, and separation of the proteins to yield the products.34 ± 730 ± 331.1a The values for kIII and k-III, obtained at pH 7.5, are similar to those obtained previously at pH 6.5 (11Meadow N.D. Roseman S. J. Biol. Chem. 1996; 271: 33440-33445Abstract Full Text Full Text PDF PubMed Scopus (21) Google Scholar), which were 61 × 106 m-1 s-1 and 47 × 106 m-1 s-1, respectively.b One of the experiments with this protein was started in the reverse direction, i.e., with HPr as the acceptor and [32P]phospho-IIAGlc as the donor. The rate constants derived from experiments started from opposite directions were in good agreement, indicating that there are no significant concentrations of intermediate complexes between the two reacting proteins prior to the last step, transfer of the phosphoryl group to the acceptor, and separation of the proteins to yield the products. Open table in a new tab The rate constants of the T73A and T73S mutant proteins are reduced by a factor of less than two relative to those of the wild-type protein. The T73V mutant shows a somewhat larger defect, about a factor of three. These reductions obviously do not explain the 200-fold effect observed the H75Q mutation. Thus, the present results suggest that the hydrogen bond between Thr73 and His90 is not catalytic. It therefore appears likely that the 200-fold decrease in the H75Q mutation ensues from loss of transition state stabilization by the imidazole ring of His75 and that this function cannot be effectively replaced by the amido group of glutamine. In the PTS reaction sequence (Fig. 1), IIAGlc accepts a phosphoryl group from [P]HPr and is a donor to the IIB (cytoplasmic) domain of the membrane protein, IICBGlc. The reactions studied here with the cloned, soluble IIBGlc domain are referred to as Reaction IVa in Scheme 1. We previously reported that proteolysis of the seven N-terminal residues of IIAGlc from S. typhimurium reduced its activity in sugar phosphorylation assays to a few percent of the activity of the full-length protein (13Meadow N.D. Roseman S. J. Biol. Chem. 1982; 257: 14526-14537Abstract Full Text PDF PubMed Google Scholar). In the present studies, we test the role of the N-terminal domain of IIAGlc on its kinetic properties by constructing and assaying two truncations, (Δ7)IIAGlc and (Δ16)IIAGlc, as described under “Experimental Procedures.” The phosphotransfer properties of the truncations were tested with the soluble proteins, HPr and IIBGlc, and with the membrane protein IICBGlc. A differential effect on the rate constants might indicate whether the N-terminal domain requires membrane lipid to exert its effect. Effects of Truncations on Phosphotransfer Reactions with HPr—Experimental and theoretical progress curves for phosphotransfer Reaction III between (Δ7)IIAGlc and HPr are shown in Fig. 3, and a summary of six experiments is given in Table 1. Truncation has only a small effect on Reaction III, reducing both kIII and k–III by a factor of about two, and the length of the truncation did not matter. Two recent studies of the interacting complexes of HPr and IIAGlc (34Wang G. Louis J.M. Sondej M. Seok Y.-J. Peterkofsky A. Clore G.M. EMBO J. 2000; 19: 5635-5649Crossref PubMed Scopus (106) Google Scholar) and IIAGlc and IIBGlc (35Cai M. Williams Jr., D.C. Wang G. Lee B.R. Peterkofsky A. Clore G.M. J. Biol. Chem. 2003; 278: 25191-25206Abstract Full Text Full Text PDF PubMed Scopus (61) Google Scholar) have shown that the N-terminal domain of IIAGlc remains unstructured in both interactions. The weak effects of truncation on the kinetics of phosphotransfer are consistent with these observations. Effects of Truncations on Reactions with IIBGlc6His—A progress curve of P-transfer between the truncated IIAGlc proteins and IIBGlc is given in Fig. 4, and the rate constants for this reaction are summarized in Table 2. Both truncations resulted in an increased rate constant for P-transfer, i.e. a small effect, ∼1.4-fold, in the forward direction, and ∼6-fold in the reverse reaction (i.e. Reaction IVa).TABLE 2Rate constants for phosphotransfer between IIAGlc mutants and IIBGlc6HisIIAGlckIVa(kIV)aThe values in parentheses are for kIV and k-IV.k-IVa(k-IV)aThe values in parentheses are for kIV and k-IV.nKeq'm-1 s-1 × 10-6wt + IICBGlc(3.7)aThe values in parentheses are for kIV and k-IV., bThese data are from Ref. 28 and are shown here so that they can be compared to the data with the cloned domain.(0.3)aThe values in parentheses are for kIV and k-IV., bThese data are from Ref. 28 and are shown here so that they can be compared to the data with the cloned domain.12wt + IIBGlc8.0 ± 0.3bThese data are from Ref. 28 and are shown here so that they can be compared to the data with the cloned domain.2.3 ± 0.24bThese data are from Ref. 28 and are shown here so that they can be compared to the data with the cloned domain.3.5Δ7 + IIBGlc10 ± 0.714 ± 220.7Δ16 + IIBGlc121510.8a The values in parentheses are for kIV and k-IV.b These data are from Ref. 28Meadow N.D. Savtchenko R.S. Nezami A. Roseman S. J. Biol. Chem. 2005; 280: 41872-41880Abstract Full Text Full Text PDF PubMed Scopus (8) Google Scholar and are shown here so that they can be compared to the data with the cloned domain. Open table in a new tab There is no simple structural interpretation of these differing results: the decreased rate constants with HPr and the increased rate constants of the truncated mutants with IIBGlc. However, the results suggest that the N-terminal domain of IIAGlc can interact weakly IIBGlc, even at the low concentrations of the proteins used for these experiments. Effects of Truncations on Sugar Phosphorylation Rates in the Complete PTS System; Steady-state Assays—Proteolytic truncation of IIAGlc reduced its activity in sugar phosphorylation assays by ∼30-fold (11Meadow N.D. Roseman S. J. Biol. Chem. 1996; 271: 33440-33445Abstract Full Text Full Text PDF PubMed Scopus (21) Google Scholar). The earlier experiments employed a partially purified preparation of IICBGlc (27Waygood E.B. Meadow N.D. Roseman S. Anal. Biochem. 1979; 95: 293-304Crossref PubMed Scopus (66) Google Scholar) to assay homogeneous, intact, or truncated IIAGlc, all from S. typhimurium, whereas all the work presented here employed proteins from E. coli. To clarify this issue, we performed sugar phosphorylation assays with the genetically modified (Δ7)IIAGlc (E. coli) and IICBGlc from both E. coli and S. typhimurium. The effects of truncation of E. coli IIAGlc on the specificity constants of IICBGlc from E. coli and S. typhimurium are shown in Table 3.TABLE 3Specificity constants for phosphotransfer between IIAGlc, wild type, and mutants, and IICBGlc from E. coli and S. typhimuriumIIAGlckcat/Km([P]IIAGlc)nm-1 s-1 × 10-6wt + IICBGlc (E. coli)3.8 ± 1.42wt + IICBGlc (S. typhimurium)3.1 ± 1.82Δ7 + IICBGlc (E. coli)0.97 ± 0.232Δ7 + IICBGlc (S. typhimurium)0.26 ± 0.182 Open table in a new tab Truncation of IIAGlc reduces the specificity constant of E. coli IICBGlc by a factor of about 4 relative to intact IIAGlc, whereas the specificity constant of S. typhimurium IICBGlc is reduced by a factor of ∼12, in reasonable agreement with the previously published results. The 4-fold change with E. coli membranes may seem small, but may be very physiologically significant. In a previous paper (38Rohwer J.M. Meadow N.D. Roseman S. Westerhoff H.V. Postma P.W. J. Biol. Chem. 2000; 275: 34909-34921Abstract Full Text Full Text PDF PubMed Scopus (110) Google Scholar) we report the results of transport experiments with whole cell and found that the pivotal protein in Glc transport was IICBGlc at its usual cellular levels. As can be seen in Fig. 1 of that paper (panel d), decreasing the activity of the Glc transporter by a factor of 4 would reduce the Glc uptake rate to close to 0, certainly enough to seriously affect growth. The differences in the effect of truncation of IIAGlc on its activity with E. coli and S. typhimurium IICBGlc membrane preparations could be an anomaly resulting, for instance, from differences in the physical structures of the two membrane preparations, or, could be more significant, reflecting differences in the phospholipid compositions surrounding the Glc transport proteins, or, of the intrinsic properties of the proteins. 3It should be noted that the predicted amino acid sequences of IIAGlc from the two species are virtually identical, differing in only three residues (two are Ile for Val replacements, but one is a Pro (S. typhimurium) for Thr (E. coli) replacement). The primary sequences of the IICBGlc proteins are also very similar; a Blast comparison (36Altschul S.F. Madden T.L. Schaffer A.A. Zhang J. Zhang Z. Miller W. Lipman D.J. Nucleic Acids Res. 1997; 25: 3389-3402Crossref PubMed Scopus (59926) Google Scholar) showed 97% identity; only one of the differences produces a change in a charged residue, the replacement of Met249 in S. typhimurium with Lys in E. coli. There are, however, marked differences between the proteins from the two species. The reported isoelectric points for the “homogeneous” IICBGlc proteins are pH 6.5 for the S. typhimurium protein (29Bouma C.L. Meadow N.D. Stover E.W. Roseman S. Proc. Natl. Acad. Sci., U. S. A. 1987; 84: 930-934Crossref PubMed Scopus (23) Google Scholar) and pH 9.0 for the E. coli IICBGlc (37Meins M. Zanolari B. Rosenbusch J.P. Erni B. J. Biol. Chem. 1988; 263: 12986-12993Abstract Full Text PDF PubMed Google Scholar), and the proteins react differently to antisera raised against the S. typhimurium protein (37Meins M. Zanolari B. Rosenbusch J.P. Erni B. J. Biol. Chem. 1988; 263: 12986-12993Abstract Full Text PDF PubMed Google Scholar). These results suggest a significant difference in folding of the two polypeptide chains, despite the great similarity in the amino acid sequences. Conclusions—The N-terminal domain (first 18 amino acids) of IIAGlc has been shown to be unstructured both in crystallographic and NMR experiments (6Worthylake D. Meadow N.D. Roseman S. Liao D.-I. Herzberg O. Remington S.J. Proc. Natl. Acad. Sci. U. S. A. 1991; 88: 10382-10386Crossref PubMed Scopus (88) Google Scholar, 7Feese M.D. Comolli L. Meadow N.D. Roseman S. Remington S.J. Biochemistry. 1997; 36: 16087-16096Crossref PubMed Scopus (35) Google Scholar, 8Pelton J.G. Torchia D.A. Meadow N.D. Wong C.-Y. Roseman S. Proc. Natl. Acad. Sci., U. S. A. 1991; 88: 3479-3483Crossref PubMed Scopus (38) Google Scholar), and this domain is not located near the active site His90. Thus, there was no obvious explanation for the substantial loss in activity when IIAGlc was truncated (N-terminal 7 amino acids) by a specific membrane protease (15Meadow N.D. Coyle P. Komoryia A. Anfinsen C.B. Roseman S. J. Biol. Chem. 1986; 261: 13504-13509Abstract Full Text PDF PubMed Google Scholar). The results reported here with genetically engineered Δ7 and Δ16 amino acid truncations explicitly show that the N terminus has no large effect on phosphotransfer to and from the soluble proteins, HPr and IIBGlc, but there is suggestive evidence for a weak interaction with IIBGlc. This observation is important because IIBGlc is a domain of the membrane protein, IICBGlc, that is affected more strongly by the truncations. Our results can be explained and provide support for the contention by Wang et al. (16Wang G. Keifer P.A. Peterkofsky A. Protein Sci. 2003; 12: 1087-1096Crossref PubMed Scopus (46) Google Scholar, 17Wang G. Peterkofsky A. Clore G.M. J. Biol. Chem. 2000; 275: 39811-39814Abstract Full Text Full Text PDF PubMed Scopus (44) Google Scholar) that the N-terminal domain forms an amphipathic helix in the presence of anionic phospholipids and their proposal that this helix enhances the interaction of [P]IIAGlc and IICBGlc during phosphotransfer. In other words, that the N-terminal domain acts in “docking” of phospho-IIAGlc close to the active site Cys of the IIBGlc domain of the membrane protein IICBGlc. But for the N-terminal domain to specifically direct this attachment in the correct position on a membrane containing innumerable proteins requires somewhat more than a nonspecific amphipathic helix. The kinetic results, although relatively small, suggest in fact that the N-terminal domain does play a role in the interaction between P-IIAGlc and IIBGlc. Perhaps the large effect observed with the truncated IIAGlc in the PTS sugar phosphorylation assay, especially with the S. typhimurium membranes, reflects loss of a synergistic effect in the IIAGlc truncations. That is, the role of the N-terminal domain is to form an amphipathic helix that binds to the lipid bilayer and to interact with the IIBGlc domain in IICBGlc. We thank Drs. Dimitri Toptygin and Ludwig Brand for advice on computer modeling of kinetic data.