Characterization of the Soluble Domain of the ABC7 Type Transporter Atm1
2003; Elsevier BV; Volume: 278; Issue: 52 Linguagem: Inglês
10.1074/jbc.m306472200
ISSN1083-351X
Autores Tópico(s)Metalloenzymes and iron-sulfur proteins
ResumoAtm1 is an ABC transporter that is located in yeast mitochondria and has previously been implicated in the maturation of cytosolic iron-sulfur cluster proteins. The soluble nucleotide binding domain of Atm1 (Atm1-C) has been overexpressed in Escherichia coli, purified, and characterized. Dissociation constants (KD) for Atm1-C binding of ATP (KD ∼97 μm, pH 7.3, and ∼102 μm, pH 10.0) and ADP (KD ∼43 μm, pH 7.3, and 92 μm, pH 10.0) were measured by fluorimetry. The higher binding affinity for ADP suggests that the transmembrane-spanning domain may be required to promote a structural change in the nucleotide binding domain to facilitate substrate export and ADP release. ADP also had an inhibitory effect on Atm1-C with an IC50 of 10 mm. The Michaelis-Menten constants Vmax, KM, and kcat of Atm1-C were measured as 1.822 μm min–1, 513 μm, and 0.055 min–1, respectively. The metal dependence of Atm1-C ATPase demonstrated a reactivity order of Mn2+ > Mg2+ > Co2+, while Mg2+ and Co2+ were both found to be inhibitory at higher concentrations. The pH profile and structural comparison with HisP are consistent with a role for His and Lys in promoting the ATPase activity. Structural analysis of Atm1-C by CD spectroscopy suggested a similarity of secondary structure to that found for a prokaryotic homologue (HisP), whereas modeling of the Atm1-C tertiary structure using HisP as a template is also consistent with a similarity in tertiary structure. Atm1-C tends to form a dimer or higher aggregation state at higher concentration; however, the concentration dependence of Atm1-C on ATPase activity and the results of a Hill analysis (napp = 1.1) demonstrated that there was essentially no cooperativity in ATP hydrolysis, in contrast to observations for the prokaryotic HisP transporter, which demonstrated full cooperativity for both full-length and the soluble domains. Accordingly, any cooperative response must be mediated through the transmembrane domain in the case of the eukaryotic Atm1 transporter. Atm1 is an ABC transporter that is located in yeast mitochondria and has previously been implicated in the maturation of cytosolic iron-sulfur cluster proteins. The soluble nucleotide binding domain of Atm1 (Atm1-C) has been overexpressed in Escherichia coli, purified, and characterized. Dissociation constants (KD) for Atm1-C binding of ATP (KD ∼97 μm, pH 7.3, and ∼102 μm, pH 10.0) and ADP (KD ∼43 μm, pH 7.3, and 92 μm, pH 10.0) were measured by fluorimetry. The higher binding affinity for ADP suggests that the transmembrane-spanning domain may be required to promote a structural change in the nucleotide binding domain to facilitate substrate export and ADP release. ADP also had an inhibitory effect on Atm1-C with an IC50 of 10 mm. The Michaelis-Menten constants Vmax, KM, and kcat of Atm1-C were measured as 1.822 μm min–1, 513 μm, and 0.055 min–1, respectively. The metal dependence of Atm1-C ATPase demonstrated a reactivity order of Mn2+ > Mg2+ > Co2+, while Mg2+ and Co2+ were both found to be inhibitory at higher concentrations. The pH profile and structural comparison with HisP are consistent with a role for His and Lys in promoting the ATPase activity. Structural analysis of Atm1-C by CD spectroscopy suggested a similarity of secondary structure to that found for a prokaryotic homologue (HisP), whereas modeling of the Atm1-C tertiary structure using HisP as a template is also consistent with a similarity in tertiary structure. Atm1-C tends to form a dimer or higher aggregation state at higher concentration; however, the concentration dependence of Atm1-C on ATPase activity and the results of a Hill analysis (napp = 1.1) demonstrated that there was essentially no cooperativity in ATP hydrolysis, in contrast to observations for the prokaryotic HisP transporter, which demonstrated full cooperativity for both full-length and the soluble domains. Accordingly, any cooperative response must be mediated through the transmembrane domain in the case of the eukaryotic Atm1 transporter. The ATP binding cassette (ABC) 1The abbreviations used are: ABCATP-binding cassetteFPLCfast protein purification liquid chromatographyHisPhistidine permeaseNBDnucleotide-binding domain. transporters comprise a large family of integral membrane proteins responsible for the ATP-dependent translocation of solutes across biological membranes in both prokaryotes and eukaryotes. ABC transporters are composed of four structural domains: two nucleotide-binding domains (NBDs), which show a high degree of sequence similarity throughout the family, and two transmembrane domains that typically show six transmembrane-spanning helices (1Schneider E. Hunke S. FEMS Microbiol. Rev. 1998; 22: 1-20Crossref PubMed Google Scholar). The structure and function of ABC-type proteins have been intensively studied (2Jones P.M. George A.M. FEMS Microbiol. Lett. 1999; 179: 187-202Crossref PubMed Google Scholar, 3Braibant M. Gilot P. Content J. FEMS Microbiol. Rev. 2000; 24: 449-467Crossref PubMed Google Scholar, 4Holland I.B. Blight M.A. J. Mol. Biol. 1999; 293: 381-399Crossref PubMed Scopus (489) Google Scholar, 5Higgins C.F. Res. Microbiol. 2001; 152: 205-210Crossref PubMed Scopus (481) Google Scholar, 6Koster W. Res. Microbiol. 2001; 152: 291-301Crossref PubMed Scopus (212) Google Scholar). Recently, the first high resolution x-ray crystal structure of a prokaryotic NBD of histidine permease (HisP) was reported (7Hung L.-W. Wang I.X. Nikaido K. Liu P.-Q. Ames G.F.-L. Kim S.-H. Nature. 1998; 396: 703-707Crossref PubMed Scopus (619) Google Scholar) and has provided insight on the structural basis for function in this class of ATP-dependent transporters. In prokaryotic systems, these transport complexes are usually constituted of individual subunits, whereas in the eukaryotic ABC transporters, they are normally formed from a single peptide chain (1Schneider E. Hunke S. FEMS Microbiol. Rev. 1998; 22: 1-20Crossref PubMed Google Scholar), so a comparison of prokaryotic and eukaryotic systems may yield considerable insight into structure-function correlations between these discrete families. ATP-binding cassette fast protein purification liquid chromatography histidine permease nucleotide-binding domain. Saccharomyces cerevisiae Atm1p is an ABC transporter that is located in the mitochondrial inner membrane and has been implicated in iron-sulfur cluster assembly and maturation in the cytosol (8Leighton J. Schatz G. EMBO J. 1995; 14: 188-195Crossref PubMed Scopus (170) Google Scholar, 9Kispal G. Csere P. Guiard B. Lill R. FEBS Lett. 1997; 418: 346-350Crossref PubMed Scopus (247) Google Scholar, 10Lill R. Kispal G. Trends Biochem. Sci. 2000; 25: 352-356Abstract Full Text Full Text PDF PubMed Scopus (319) Google Scholar, 11Lill R. Kispal G. Res. Microbiol. 2001; 152: 331-340Crossref PubMed Scopus (63) Google Scholar, 12Muhlenhoff U. Lill R. Biochim. Biophys. Acta. 2000; 1459: 370-382Crossref PubMed Scopus (181) Google Scholar, 13Schueck N.D. Woontner M. Koeller D.M. Mitochondrion. 2001; 1: 51-60Crossref PubMed Scopus (30) Google Scholar). It is a transmembrane-spanning protein that is putatively involved in the transfer of iron-sulfur clusters from the mitochondrial matrix to the cytosol (14Kispal G. Csere P. Prohl C. Lill R. EMBO J. 1999; 18: 3981-3989Crossref PubMed Scopus (589) Google Scholar). In addition to the transmembrane-spanning domain, the protein also possesses a soluble nucleotide-binding domain that mediates hydrolysis of ATP to ADP. Presumably, this ATPase activity is involved in regulating the opening and closing of the channel and/or driving substrate through the channel. The human homologue of Atm1p, ABC7, displays a very high sequence homology in the soluble nucleotide binding domain. Defects in the human ABC7 gene have been shown to cause a rare type of X-linked sideroblastic anemia associated with cerebellar ataxia (XLSA/A) (15Shimada Y. Okuno S. Kawai A. Shinomiya H. Saito A. Suzuki M. Omori Y. Nishino N. Kanemoto N. Fujiwara T. Horie M. Takahashi E.-I. J. Hum. Genet. 1998; 43: 115-122Crossref PubMed Scopus (92) Google Scholar, 16Allikmets R. Raskind W.H. Hutchinson A. Schueck N.D. Dean M. Koeller D.M. Hum. Mol. Genet. 1999; 8: 743-749Crossref PubMed Scopus (353) Google Scholar, 17Bekri S. Kispal G. Lange H. Fitzsimons E. Tolmie J. Lill R. Bishop D.F. Blood. 2000; 96: 3256-3264Crossref PubMed Google Scholar, 18Maguire A. Hellier K. Hammans S. May A. Br. J. Haematol. 2001; 115: 910-917Crossref PubMed Scopus (62) Google Scholar, 19Csere P. Lill R. Kispal G. FEBS Lett. 1998; 441: 266-270Crossref PubMed Scopus (120) Google Scholar). Similar mutations in Atm1p or ABC7 have been shown to result in the accumulation of high levels of free iron in the mitochondrion and a lack of iron-sulfur clusters in cytosolic proteins. Three other proteins (Bat1, Bat2, and Erv1) have also been suggested to assist this ABC transporter for iron-sulfur cluster translocation (20Kispal G. Steiner H. Court D.A. Rolinski B. Lill R. J. Biol. Chem. 1996; 271: 24458-24464Abstract Full Text Full Text PDF PubMed Scopus (122) Google Scholar, 21Lange H. Lisowsky T. Gerber J. Muhlenhoff U. Kispal G. Lill R. EMBO Rep. 2001; 2: 715-720Crossref PubMed Scopus (248) Google Scholar); however, the functional role of Atm1p in mediating iron-sulfur cluster trafficking remains unknown. Three mitochondrial ABC transporters have thus far been discovered in yeast (11Lill R. Kispal G. Res. Microbiol. 2001; 152: 331-340Crossref PubMed Scopus (63) Google Scholar), although the structural and functional properties of these eukaryotic transporters are poorly understood. In this paper, we report the cloning, purification, and characterization of the soluble C-terminal ATP-binding domain of the Schizosaccharomyces pombe Atm1 (Atm1-C) and make a detailed comparison with the bacterial HisP protein. This provides a starting point for studies of eukaryotic ABC7-type transporters and an initial step toward the understanding of iron-sulfur cluster export mechanisms in mitochondria. Construction of pET21-Atm1-C—The soluble C-terminal domain of Atm1 (Atm1-C) was predicted by computer analysis of the transmembrane segment using the program Dense Alignment Surface Method (22Cserzo M. Wallin E. Simon I. Von Heijne G. Elofsson A. Protein Eng. 1997; 10: 673-676Crossref PubMed Google Scholar). The nucleotide-binding domain of the prokaryotic HisP was found to be similar to that for Atm1-C and was further characterized by use of the search tool SWISS-MODEL (available on the World Wide Web at www.expasy.org/swissmod/SWISS-MODEL.html). The Atm1-C sequence starts from Leu436 of Atm1, and the corresponding gene fragment encoding Atm1-C was PCR-amplified by use of the following primers: 5′-GGC CGC CAT ATG CTT AAA GGC GGA TCT ATT CAA TTC G-3′ and 5′-CGC GGA TCC TCA TGC ATC TCC GGA TTT ATT CGA TTC-3′. Subsequently, this gene was cloned into pET-21b(+) between the NdeI and BamHI restriction sites. The sequence of expression vector pET21-Atm1-C was finally confirmed by DNA sequence analysis and subsequently transformed into an E. coli BL21(DE3) host. Overexpression and Purification of Atm1-C—Expression plasmid pET21-Atm1-C was transformed into the expression host E. coli BL21(DE3) as described elsewhere (23Maniatis T. Fritsch E.F. Sambrook J. Molecular Cloning: A Laboratory Manual. 2nd Ed. Cold Spring Harbor Laboratory, Cold Spring Harbor, NY1989: 1.82-1.84Google Scholar). Cells were grown to an A600 ∼0.7 and were then induced with 0.1 mm isopropyl-1-thio-β-d-galactopyranoside at 37 °C for 4 h. After harvesting and washing with 50 mm Tris-HCl, pH 7.3, the cells were lysed by sonication with 50 ml of 50 mm Tris-HCl, pH 7.3. Since Atm1-C was found to form inclusion bodies, following sonication, the pellet was washed in turn with 50 ml of each of 50 mm Tris-HCl, 50 mm Tris-HCl plus 100 mm NaCl, 50 mm Tris-HCl plus 200 mm NaCl, 50 mm Tris-HCl plus 500 mm NaCl, and 50 mm Tris-HCl plus 1% Triton X-100, pH 7.3, buffers to remove soluble and membrane-associated proteins. Subsequently, the Atm1-C inclusion body was dissolved in 20 ml of solubilization buffer (50 mm Tris-HCl, 100 mm NaCl, 2 m guanidine HCl, 1 mm ATP, 0.1 mm EDTA, pH 10.0) at 4 °C overnight. The denatured solution was centrifuged at 15,000 rpm to remove the insoluble pellet. Subsequently, the supernatant was diluted 10-fold by the slow addition of a pH 10.0 buffer containing 50 mm Tris-HCl, 100 mm NaCl, 1 mm ATP, and 0.1 mm EDTA at 4 °C. After renaturation, the refolded Atm1-C was concentrated by Amicon ultrafiltration. Pure Atm1-C was obtained following ion exchange chromatography (DE52) and gel filtration (G75) chromatography. For DE52 purification, the column was washed with 500 ml of 50 mm Tris-OH, pH 10.0, and subsequently the protein was eluted with a gradient from 0 m to 250 mm NaCl in 50 mm Tris-OH and 0.1 mm EDTA, pH 10.0. The running conditions for the G75 column included an equilibration buffer with 50 mm Tris-OH, 100 mm NaCl, and 0.1 mm EDTA, pH 10.0. The purity of Atm1-C was based on 12% SDS-PAGE analysis. Determination of the Aggregation State of Atm1-C—The aggregation state of Atm1-C was determined by gel filtration chromatography using a Superose 12 column on an Amersham Biosciences FPLC system. The experiment was performed in a pH 10.0 buffer containing 100 mm Tris-HCl, 100 mm NaCl, and 0.1 mm EDTA. The gel filtration column had previously been calibrated with the FPLC molecular mass standards lysozyme (14.3 kDa), deoxyribonuclease I (31 kDa), pepsin (35 kDa), bovine serum albumin (66 kDa), and bovine serum albumin dimer (132 kDa). Blue dextrin was used to determine the dead volume. Molecular masses were determined by plotting log molecular masses of standards versus Kav, where Kav = (Ve – V0)/(Vt – V0), Ve represents elution volume, V0 is dead volume, and Vt is total column volume. Fluorescence Determination of ATP and ADP Binding Constant to Atm1-C by Fluorimetry—A 500-μl solution of 10 μm Atm1-C in 50 mm Tris-HCl (pH 7.3 or pH 10.0) was titrated with 1-μl aliquots of ATP or ADP (from 5 or 50 mm stocks) in reaction buffers containing 50 mm Tris-HCl (pH 7.3 or 10.0). The total volume of titrant added was 20 μl, and the effect of the dilution on the emission intensity was negligible. The change in fluorescence emission was monitored by use of a PerkinElmer LS50B luminescence spectrometer with an excitation wavelength of 280 nm (Tyr and Trp excitation) or 295 nm (Trp excitation) and an emission wavelength of 330 or 340 nm, respectively. The data were plotted as ΔI/Io versus ATP or ADP concentration and fitted by use of the equation ΔI/Io = constant[S]/(KD + [S]) (24Encinas M.V. Rojas M.C. Goldie H. Cardemil E. Biochim. Biophys. Acta. 1993; 1162: 195-202Crossref PubMed Scopus (21) Google Scholar), where ΔI = (Io – Iobs) and Iobs is the observed emission following each addition of titrant with a resulting nucleotide concentration defined by [S]. The constant accounts for the fact that the saturating emission is not necessarily zero (i.e. the emission is not completely quenched). Corrections for the inner filter effect were made using the equation Icorr = Iobs·10A/2 (25Lehrer S.S. Leavis P.C. Methods Enzymol. 1978; 49: 4406-4412Google Scholar), where Icorr represents the corrected emission intensity, Iobs is the observed intensity, and A is the absorbance at the excitation wave-length. This accounted for any change in emission intensity that results from nonbinding events such as absorption of light by the nucleotide, although such corrections were found to be minor. To confirm the requirement for a structured binding pocket to promote nucleotide binding, a control experiment was carried out. A mixture containing 150 μl of 10 μm Atm1-C in 50 mm Tris-HCl and 6 m guanidine HCl, pH 7.3, was titrated with ATP or ADP in reaction buffer 50 mm Tris-HCl and 6 m guanidine HCl, pH 7.3, and the variation in emission intensity was monitored at 330 and 340 nm as described earlier. ATPase Activity Assay—A 350-μl volume of a reaction mixture containing 30 μm of Atm1-C in an assay buffer consisting of 50 mm Tris-HCl, 100 mm NaCl, and 0.1 mm EDTA (pH 7.3 or 10.0) was incubated with 2 mm ATP at 37 °C for 3 min, and the reaction was initiated by the addition of 50 mm MgCl2 to a final concentration 2 mm. A 50-μl aliquot of the reaction mixture was taken and placed in a tube containing 50 μl of 7.5% SDS. The amount of inorganic phosphate released was determined by a colorimetric assay, using Na2HPO4 as a standard and monitoring the absorbance change at 820 nm (26Nikaido K. Liu P.-Q. Ames G.F.-L. J. Biol. Chem. 1997; 272: 27745-27752Abstract Full Text Full Text PDF PubMed Scopus (105) Google Scholar, 27Chifflet S. Torriglia A. Chiesa R. Tolosa S. Anal. Biochem. 1988; 168: 1-4Crossref PubMed Scopus (418) Google Scholar). The pH profile for the reaction displayed bell-shaped behavior and was fit by use of Equation 1, where Vmax represents the maximal activity under saturating conditions, Vopt represents the optimum initial velocity with respect to pH, and the pKa values represent the fitted ionization constants assuming two ionization events.Vmax=Vopt/(1+10-pH/10-pKa1+10-pKa2/10-pH)(Eq. 1) Influence of Phospholipid on ATPase Activity—Experiments to examine the phospholipid dependence of the reaction of Atm1-C were performed with 20 μm Atm1-C with 2 mm ATP in a pH 8.0 assay buffer containing 50 mm Tris-HCl, 100 mm NaCl, and 0.1 mm EDTA. The reaction was preincubated on ice for 30 min with 0.04, 0.2, and 1 mg/ml phosphatidylethanolamine or cardiolipin and initiated by the addition of MgCl2 to a final concentration of 2 mm. Quantitation of the amount and rate of phosphate release has been described under “ATPase Activity Assay.” Metal Dependence of the ATPase Activity of Atm1-C—The metal dependence of the reaction was examined with 20 μm Atm1-C and 2 mm ATP in an assay buffer containing 50 mm Tris-HCl, pH 8.0, 100 mm NaCl, and 0.1 mm EDTA. The reaction was initiated by the addition of Mg2+, Co2+, or Mn2+ to the desired concentration. Quantitation of the amount and rate of phosphate release has been described under “ATPase Activity Assay.” For Mg2+ and Co2+, which displayed inhibitory effects at higher [M2+], Equation 2 was used, whereas the simpler Equation 3 was used for Mn2+ that showed no inhibitory behavior, where kcat and KM are the standard Michaelis constants, KM2+ is the dissociation constant for metal promoted activity, KI is the inhibition constant, and other concentration terms are total enzyme, substrate, and metal ion concentrations. Equations 2 and 3 were readily derived from standard equations (28Fresht A. Enzyme Kinetics,2nd Ed. W. H. Freeman and Co., New York1997: 107-109Google Scholar) by assuming a bound metal to be required for activity and a bound metal requirement for inhibition.V0=kcat·[Eo]·[So]·KI·KM2+·[Mg2+]/((1+KM2+·[Mg2+])·(KI·([S∘]+KM)+KM·[Mg2+]))(Eq. 2) V0=kcat·[Eo]·[So]·KM2+·[Mg2+]/((1+KM2+·[Mg2+])·([So]+KM))(Eq. 3) Circular Dichroism—Circular dichroism spectra were measured on an Aviv model 202 circular dichroism spectrometer. Far-UV CD spectra were acquired with a 0.3-mm path length cuvette. Concentration of Atm1-C was 10 μm in 20 mm potassium phosphate, pH 7.4, and 100 mm KCl. Spectra acquired at 20 °C were determined per 0.2 nm in triplicate and averaged. Secondary structure quantitation was determined via the self-consistent method (29Sreerama N. Woody R.W. J. Mol. Biol. 1994; 242: 497-507PubMed Google Scholar) with the Dicroprot version 2.5 version 5.0 package (30Deleage G. Geourjon C. Comput. Appl. Biosci. 1993; 9: 197-199PubMed Google Scholar) obtained from the Institut de Biologie et Chimie des Protéines (available on the World Wide Web at www.ibcp.fr). Buffer spectra were always subtracted. Modeling—The three-dimensional structure of Atm1-C was predicted by use of the SwissModel First Approach Mode. The input sequence of Atm1-C starts from Leu436 of Atm1 with an additional Met at the N terminus. The lower Blast P(N) limit for template selection was set to 0.00001. The three-dimensional structure of HisP was also used as the self-input template file for the tertiary structure prediction of Atm1-C. The final model output was a Swiss-PDB viewer project file (31Guex N. Peitsch M.C. Electrophoresis. 1997; 18: 2714-2723Crossref PubMed Scopus (9634) Google Scholar, 32Peitsch M.C. BioTechnology. 1995; 13: 658-660Crossref Scopus (116) Google Scholar, 33Schwede T. Kopp J. Guex N. Peitsch M.C. Nucleic Acids Res. 2003; 31: 3381-3385Crossref PubMed Scopus (4530) Google Scholar). Design and Cloning of the Truncated Atm1-C Gene—The amino acid sequence designation of the C-terminal soluble domain of Atm1, Atm1-C, was based both on the use of a tool to search for similar sequences (SWISS-MODEL) and on the structure of the sequence-related bacterial protein, HisP. The amino acid comparison of HisP and Atm1-C is shown in Fig. 1 (34Notredame C. Higgins D.G. Heringa J. J. Mol. Biol. 2000; 302: 205-217Crossref PubMed Scopus (5507) Google Scholar), and the predicted transmembrane-spanning region (22Cserzo M. Wallin E. Simon I. Von Heijne G. Elofsson A. Protein Eng. 1997; 10: 673-676Crossref PubMed Google Scholar) also provided important guidelines for construction of the nucleotide-binding domain of Atm1. Structural information is limited for ABC transporters, and HisP is the only representative of this family with a high resolution structure for the nucleotide-binding domain (7Hung L.-W. Wang I.X. Nikaido K. Liu P.-Q. Ames G.F.-L. Kim S.-H. Nature. 1998; 396: 703-707Crossref PubMed Scopus (619) Google Scholar). Characterization of Atm1-C Aggregation State—Purified Atm1-C was observed to form a white flurry during concentration by Amicon ultrafiltration, especially in the vicinity of the membrane surface. This most likely reflects contact by a membrane-associated domain remaining in this protein. The aggregation state of Atm1-C was determined by use of gel filtration chromatography. The FPLC elution profile (Fig. 2) showed that most of the expressed Atm1-C was formed as a high molecular weight aggregate with a small amount of dimer. A very low concentration of monomer was also detected in buffer lacking β-mercaptoethanol during gel filtration chromatography. However, Fig. 2 shows that in buffer containing 0.5% β-mercaptoethanol, the relative amounts of both the high molecular weight aggregate and the dimeric form were shown to dramatically decrease relative to the amount of monomer. This suggested formation of intermolecular disulfide bonds in Atm1-C as the principal reason for formation of the high molecular weight aggregate and dimer. By use of appropriate standards, the molecular mass of monomer was determined by gel filtration as 28.5 kDa, and the molecular mass of dimer was determined as 56.9 kDa. These are similar to the calculated masses of 26.9 and 58.9 kDa, respectively. The molecular mass of each eluted fraction was confirmed by SDS-PAGE. ATP and ADP Binding to Atm1-C—There are six Tyr and one Trp in the sequence of Atm1-C. Both residues are electronically excited following excitation at 280 nm, whereas only Trp is stimulated by 295-nm excitation. Nucleotide binding to Atm1-C can and does result in a decrease in the combined emission intensity of both Tyr and Trp (Fig. 3) and the measured KD values are summarized in Table I. Control experiments with denatured Atm1-C (Fig. 3) demonstrate that the change in emission intensity is a consequence of binding rather than a reduction of energy input as a result of the absorption of light by nucleotide. Specifically, the experiments show that there is no binding for either ADP or ATP after accounting for the inner filter effect of the denaturant. Since nucleotide does not absorb at 295 nm, an additional control experiment was carried out by monitoring the weaker Trp emission following 295-nm excitation (data not shown). Within error, similar binding parameters were obtained, so we conclude that the binding affinity that is measured from the change in emission intensity indeed reflects the actual binding between the refolded Atm1-C domain and ATP or ADP. It is important to note, however, that these results do not suggest a specific mechanism for quenching, whether directly through energy transfer from specific Tyr or Trp residues, or as a result of conformational changes that influence the polarity and/or solvent accessibility of the environment around such residues.Table INucleotide binding affinity to Atm1-C measured by fluorescence quenchingATPADPConstantKDConstantKDμmμmpH 7.30.60970.4343pH 10.00.481020.4892 Open table in a new tab Our results from monitoring both Tyr and Trp emission indicate that ADP binds more tightly to Atm1-C than ATP binding at both pH 10.0 and 7.3. The binding affinity at pH 10.0 is lower than that at pH 7.3, consistent with more extensive deprotonation of residues in Atm1-C at higher pH with an increase in repulsion between Atm1-C and either ATP or ADP. Atm1-C ATPase Activity and Inhibition—The ATPase activity of Atm1-C was determined from the initial rate of phosphate release, which was linear with time over a period of at least 1 h. From the resulting Michaelis-Menten plot (Fig. 4) the Vmax, KM, and kcat of Atm1-C were measured as 1.822 μm min–1 (or 0.02 μmol min–1 mg–1), 512.85 μm, and 0.0552 min–1, respectively. When analyzed by the Hill equation (35Davidson A.L. Laghaeian S.S. Mannering D.E. J. Biol. Chem. 1996; 271: 4858-4863Abstract Full Text Full Text PDF PubMed Scopus (107) Google Scholar), the apparent Hill coefficient, napp, reflecting the number of substrate binding sites per unit of active enzyme, was equal to 1.1 ± 0.2. The dependence of ATPase activity on Atm1-C concentration was determined over a range of protein concentration with 2 mm ATP substrate in each reaction. Methods for quantifying the concentration of liberated phosphate were described under “Experimental Procedures.” A linear relationship between ATPase activity and Atm1-C concentration was observed, suggesting that each subunit of Atm1-C acted as an individual unit for ATP hydrolysis and consistent with the measured Hill coefficient napp ∼1.1. The pH dependence of the ATPase activity of Atm1-C was examined between pH 5.5 and 10.0. Fig. 5 shows the optimal pH for ATP activity to be around 8, indicative of two ionization events involving distinct residues that may influence ATPase activity. The pKa values of these two residues have been determined by use of Equation 1, and the values of pKa1 and pKa2 are determined to be 7.3 and 9.3, respectively. The results are consistent with involvement of specific amino acid residues in general acid and general base catalysis of ATP hydrolysis (36Scarborough G.A. J. Bioenerg. Biomembr. 2002; 34: 235-250Crossref PubMed Scopus (28) Google Scholar). The influence of phospholipids on ATPase activity was measured by adding phosphatidylethanolamine or cardiolipin to final concentrations of 0.04, 0.2, and 1 mg/ml. Neither showed any significant effect on ATPase activity by comparison with control experiments. This result is also consistent with results from concentration dependence experiments that suggested each subunit of Atm1-C acts as an individual unit for ATP hydrolysis. Experiments to determine the inhibitory influence of several standard ATPase inhibitors were performed with 20 μm Atm1-C in the presence of 2 mm ATP. The reaction was preincubated with 2 mm inhibitor on ice for 30 min and initiated by the addition MgCl2 to a final concentration of 2 mm. Vanadate, nitrate, and azide were used to test for P-type ATPase, V-type ATPase, and F-type ATPase inhibition, respectively. None of these showed any effect on enzymatic inhibition. Only ADP had an inhibitory effect on Atm1-C with an IC50 of 10 mm. This number is comparable with that obtained for HisP (∼2 mm) (26Nikaido K. Liu P.-Q. Ames G.F.-L. J. Biol. Chem. 1997; 272: 27745-27752Abstract Full Text Full Text PDF PubMed Scopus (105) Google Scholar). Metal Cofactor Promotion and Inhibition of Atm1-C Activity— The results shown in Fig. 6 demonstrated that Mg2+ and Co2+ stimulate ATPase activity to similar extents. The optimum concentration of these two cations is 0.5 mm, although with increasing cation concentration, the ATPase activity was found to decrease. Manganous ion was found to best stimulate ATPase activity, especially at higher cation concentration. This result is generally in agreement with observations made with HisP (26Nikaido K. Liu P.-Q. Ames G.F.-L. J. Biol. Chem. 1997; 272: 27745-27752Abstract Full Text Full Text PDF PubMed Scopus (105) Google Scholar), although in the latter case each of Mg2+, Mn2+, and Co2+ were found to be inhibitory at higher concentrations, whereas only Mg2+ and Co2+ were inhibitory at higher concentrations for Atm1-C. The inhibitory influence of the metal ion dependence of ATPase activity was analyzed according to the saturation model that has been described in a previous study of metal cofactor-promoted ribonuclease H activity (37Black C.B. Cowan J.A. Inorg. Chem. 1994; 33: 5805-5808Crossref Scopus (38) Google Scholar). In this saturation model, metal ion bound substrate may become inhibitory at high metal ion concentrations, where either the nucleotide substrate or the enzyme may bind additional metal ions. For such a scenario, the metal binding constant (KM2+) and inhibition constant (KI) can be determined by Equation 2. The results are summarized in Table II, where it is seen that Co2+ shows the largest inhibitory effect on ATPase activity. Since Mn2+ shows no inhibitory influence over the concentration range used, a simpler hyperbolic binding equation (Equation 3) was used.Table IIMetal binding constants (KM2+) and inhibition constants (KI) for Atm1-C ATP hydrolysisMetal factorKM2+KImmMg2+0.51.4Co2+1.94.1Mn2+2.1 Open table
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