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• W2000955420 abstract "The potential photoaffinity probe 8-azido-adenosine (8-N3-Ado) was shown to serve as a substrate for the 3′-oxidative activity of human S-adenosylhomocysteine (AdoHcy) hydrolase (Aiyar, V. N., and Hershfield, M. S.(1985) Biochem. J. 232, 643-650). In this study, we have determined the equilibrium binding properties of 8-N3-Ado with AdoHcy hydrolase (NAD+ form) and identified the specific amino acid residues that are covalently modified. After irradiation of the reaction mixture of [2-3H]8-N3-Ado and AdoHcy hydrolase (NAD+ form) and followed by tryptic digestion, peptides specifically photolabeled by [2-3H]3′-keto-8-N3-Ado were effectively separated from peptides nonspecifically labeled with [2-3H]8-N3-Ado using boronate affinity chromatography. After purification by reverse phase high performance liquid chromatography, two photolabeled peptides were isolated and identified as Val175-Lys186 and Val319-Arg327, in which Ala177 and Ile321 were associated with radioactivity. The specificity of the photoaffinity labeling with [2-3H]3′-keto-8-N3-Ado was demonstrated by the observation that these photolabeled peptides were not isolated when [2-3H]8-N3-Ado was incubated with apo AdoHcy hydrolase and irradiated. The two photolabeled peptides are assumed to be parts of the adenine-binding domain for substrates. They are both within well conserved regions of AdoHcy hydrolases. The peptide Val175-Lys186 is located very close to Cys195 and Glu197-Ser198, both of which were indicated to be located in the active site of the enzyme by chemical modification and limited proteolysis methods. The peptide Val319-Arg327 is adjacent to Leu330, which is proposed by a computer graphics model to interact with the C-6-NH2 group of Ado. The potential photoaffinity probe 8-azido-adenosine (8-N3-Ado) was shown to serve as a substrate for the 3′-oxidative activity of human S-adenosylhomocysteine (AdoHcy) hydrolase (Aiyar, V. N., and Hershfield, M. S.(1985) Biochem. J. 232, 643-650). In this study, we have determined the equilibrium binding properties of 8-N3-Ado with AdoHcy hydrolase (NAD+ form) and identified the specific amino acid residues that are covalently modified. After irradiation of the reaction mixture of [2-3H]8-N3-Ado and AdoHcy hydrolase (NAD+ form) and followed by tryptic digestion, peptides specifically photolabeled by [2-3H]3′-keto-8-N3-Ado were effectively separated from peptides nonspecifically labeled with [2-3H]8-N3-Ado using boronate affinity chromatography. After purification by reverse phase high performance liquid chromatography, two photolabeled peptides were isolated and identified as Val175-Lys186 and Val319-Arg327, in which Ala177 and Ile321 were associated with radioactivity. The specificity of the photoaffinity labeling with [2-3H]3′-keto-8-N3-Ado was demonstrated by the observation that these photolabeled peptides were not isolated when [2-3H]8-N3-Ado was incubated with apo AdoHcy hydrolase and irradiated. The two photolabeled peptides are assumed to be parts of the adenine-binding domain for substrates. They are both within well conserved regions of AdoHcy hydrolases. The peptide Val175-Lys186 is located very close to Cys195 and Glu197-Ser198, both of which were indicated to be located in the active site of the enzyme by chemical modification and limited proteolysis methods. The peptide Val319-Arg327 is adjacent to Leu330, which is proposed by a computer graphics model to interact with the C-6-NH2 group of Ado. S-Adenosylhomocysteine (AdoHcy)1( 1The abbreviations used are: AdoHcyS-adenosyl-L-homocys-teine8-N3-Ado8-azido-adenosineHPLChigh performance liquid chromatography. 1The abbreviations used are: AdoHcyS-adenosyl-L-homocys-teine8-N3-Ado8-azido-adenosineHPLChigh performance liquid chromatography.)hydrolase (EC 3.3.1.1) catalyzes the reversible hydrolysis of AdoHcy to adenosine (Ado) and L-homocysteine (Hcy)(1De la Haba G. Cantoni G.L. J. Biol. Chem. 1959; 234: 603-608Abstract Full Text PDF PubMed Google Scholar). The mechanism of the enzyme reaction has been elegantly elucidated by Palmer and Abeles(2Palmer J.L. Abeles R.H. J. Biol. Chem. 1979; 254: 1217-1226Abstract Full Text PDF PubMed Google Scholar). AdoHcy hydrolase has been cloned from a number of different sources, including Homo sapiens(3Coulter-Karis D.E. Hershfield M.S. Ann. Hum. Genet. 1989; 53: 169-175Crossref PubMed Scopus (55) Google Scholar), Rattus species(4Ogawa H. Gomi T. Mueckler M.M. Fujioka M. Backlund Jr., P.S. Aksamit R.R. Unson C.G. Cantoni G.L. Proc. Natl. Acad. Sci. U. S. A. 1987; 84: 719-723Crossref PubMed Scopus (47) Google Scholar), Plasmodium falciparum(5Creedon K.A. Rathod P.K. Wellems T.E. J. Biol. Chem. 1994; 269: 16364-16370Abstract Full Text PDF PubMed Google Scholar), Rhodobacter capsulatus (6Sganga M.W. Aksamit R.R. Cantoni G.L. Bauer C.E. Proc. Natl. Acad. Sci. U. S. A. 1992; 89: 6328-6332Crossref PubMed Scopus (33) Google Scholar), Triticum aestivum(7Richards K.D. Gardner R.C. GenBank/EMBL accession L 11872. 1993; Google Scholar), Catharanthus roseus (8Schroeder G. Waitz A. Hotze M. Schroeder J. GenBank/EMBL accession Z 26881. 1993; Google Scholar), Petroselinum crispum(9Kawalleck P. Plesch G. Hahlbrock K. Somssich I.E. Proc. Natl. Acad. Sci. U. S. A. 1992; 89: 4713-4717Crossref PubMed Scopus (126) Google Scholar), Leishmania donovani(10Henderson D.M. Hanson S. Allen T. Wilson K. Coulter-Karis D.E. Greenberg M.L. Hershfield M.S. Ullman B. Mol. Biochem. Parasitol. 1992; 53: 169-183Crossref PubMed Scopus (57) Google Scholar), Dictyostelium discoideum(11Kasir J. Aksamit R.R. Backlund Jr., P.S. Cantoni G.L. Biochem. Biophys. Res. Commun. 1988; 153: 359-364Crossref PubMed Scopus (25) Google Scholar), and Caenorhabditis elegans(12Prasad S.S. Starr T. Rose A.M. Genome. 1993; 36: 57-65Crossref PubMed Scopus (9) Google Scholar). The amino acid sequences of the cloned AdoHcy hydrolases have been deduced from their cDNA sequences. Comparison of the amino acid sequences from these species shows a remarkable degree of conservation ranging from 64% identity between human and Rhodobacter capsulatus(6Sganga M.W. Aksamit R.R. Cantoni G.L. Bauer C.E. Proc. Natl. Acad. Sci. U. S. A. 1992; 89: 6328-6332Crossref PubMed Scopus (33) Google Scholar) to 97% identity between human and rat(3Coulter-Karis D.E. Hershfield M.S. Ann. Hum. Genet. 1989; 53: 169-175Crossref PubMed Scopus (55) Google Scholar). A highly conserved amino acid region from 213 to 244 in the recombinant rat liver enzyme has been postulated to be part of the NAD+-binding site (4Ogawa H. Gomi T. Mueckler M.M. Fujioka M. Backlund Jr., P.S. Aksamit R.R. Unson C.G. Cantoni G.L. Proc. Natl. Acad. Sci. U. S. A. 1987; 84: 719-723Crossref PubMed Scopus (47) Google Scholar) based on the fingerprint sequence of G-X-G-X-X-G, which is a characteristic βαβ-fold formed in NAD+- and FAD-binding domains of proteins that bind the ADP moiety of the dinucleotide cofactor(13Wiereng R.K. Terpstra P. Hol W.G.J. J. Mol. Biol. 1986; 187: 101-107Crossref PubMed Scopus (993) Google Scholar). Mutations of any of the 3 glycine residues produce mutant proteins that have no catalytic activity and contain no bound coenzyme(14Gomi T. Takata Y. Date T. Fujioka M. Aksamit R.R. Backlund Jr., P.S. Cantoni G.L. J. Biol. Chem. 1990; 265: 16102-16107Abstract Full Text PDF PubMed Google Scholar). S-adenosyl-L-homocys-teine 8-azido-adenosine high performance liquid chromatography. S-adenosyl-L-homocys-teine 8-azido-adenosine high performance liquid chromatography. In contrast to the coenzyme-binding site, little is known about the substrate-binding site of AdoHcy hydrolase. Efforts have been made to elucidate information about the active site residues that participate in catalysis or substrate binding using strategies including chemical modification, site-directed mutagenesis, and limited proteolysis. Two cysteine residues, 1 arginine residue, one carboxyl group, and 1 histidine residue were indicated to be essential for enzyme activity of the rat liver AdoHcy hydrolase by chemical modification studies(15Gomi T. Fujioka M. Biochemistry. 1982; 21: 4171-4176Crossref PubMed Scopus (18) Google Scholar, 16Gomi T. Fujioka M. Biochemistry. 1983; 22: 137-143Crossref PubMed Scopus (58) Google Scholar, 17Takata Y. Fujioka M. J. Biol. Chem. 1983; 258: 7374-7378Abstract Full Text PDF PubMed Google Scholar, 18Takata Y. Gomi T. Fujioka M. Arch. Biochem. Biophys. 1985; 240: 827-835Crossref PubMed Scopus (9) Google Scholar, 19Gomi T. Ogawa H. Fujioka M. J. Biol. Chem. 1986; 261: 13422-13425Abstract Full Text PDF PubMed Google Scholar). Site-directed mutagenesis studies showed that Lys426 is critical for the activity of the human enzyme and Cys78 has an important role in the structure of the rat liver enzyme(20Ault-Riché D.B. Yuan C.S. Borchardt R.T. J. Biol. Chem. 1994; 269: 31472-31478Abstract Full Text PDF PubMed Google Scholar, 21Aksamit R.R. Backlund Jr., P.S. Moos Jr., M. Caryk T. Gomi T. Ogawa H. Fujioka M. Cantoni G.L. J. Biol. Chem. 1994; 269: 4084-4091Abstract Full Text PDF PubMed Google Scholar). More recently, a limited proteolysis study demonstrated that Glu197-Ser198 is located in or near the active site of the enzyme(22Gupta R.A. Yuan C.S. Ault-Riche D.B. Borchardt R.T. Arch. Biochem. Biophys. 1995; 319: 365-371Crossref PubMed Scopus (6) Google Scholar). Another potential approach to elucidating crucial amino acids at the active site of an enzyme is photoaffinity labeling. Photoaffinity probes have been used very effectively for studying nucleotide/nucleoside-binding proteins(23Potter R.L. Haley B.E. Methods Enzymol. 1983; 91: 613-633Crossref PubMed Scopus (94) Google Scholar, 24Olcott M.C. Bradley M.L. Haley B.E. Biochemistry. 1994; 33: 11935-11941Crossref PubMed Scopus (35) Google Scholar). In earlier studies, Aiyar and Hershfield (25Aiyar V.N. Hershfield M.S. Biochem. J. 1985; 232: 643-650Crossref PubMed Scopus (11) Google Scholar) showed that 8-N3-Ado, a potential photoaffinity labeling reagent, was a substrate for the 3′-oxidative activity of AdoHcy hydrolase. In this report, we have determined the equilibrium binding properties of 8-N3-Ado with AdoHcy hydrolase (NAD+ form) and identified the specific amino acid residues that are covalently modified after irradiation of the substrate-enzyme complex. 8-N3-Ado, NAD+, NADH, and L1-tosyl-amido-2-phenylethyl chloromethyl ketone-treated trypsin (Type XIII) were purchased from Sigma. [2-3H]8-N3-Ado (17.7 Ci/mmol) was obtained from Moravek Biochemicals Inc. (Brea, CA). Boronate affinity resin Affi-Gel 601 was from Bio-Rad. Expression of the human placental AdoHcy hydrolase cDNA in Escherichia coli and purification of the enzyme were carried out as described previously(26Yuan C.S. Wnuk S.F. Liu S. Robins M.J. Borchardt R.T. Biochemistry. 1994; 33: 12305-12311Crossref PubMed Scopus (27) Google Scholar). The purified, homogeneous enzyme is a tetramer consisting of four identical subunits of Mr 47,000. In this study, the subunit Mr was used to calculate the molarity of enzyme solutions. The protein concentration was determined by the method of Bradford (27Bradford M.M. Anal. Biochem. 1976; 72: 248-254Crossref PubMed Scopus (211946) Google Scholar) using bovine serum albumin as a standard. Apo AdoHcy hydrolase was obtained by treatment of AdoHcy hydrolase (NAD+ form) with acidic (NH4)2SO4 to remove the NAD+ as described previously(28Yuan C.S. Yeh J. Liu S. Borchardt R.T. J. Biol. Chem. 1993; 268: 17030-17037Abstract Full Text PDF PubMed Google Scholar, 29Gomi T. Takata Y. Fujioka M. Biochim. Biophys. Acta. 1989; 994: 172-179Crossref PubMed Scopus (20) Google Scholar). The AdoHcy hydrolase activity was determined in the synthetic direction as described earlier (26Yuan C.S. Wnuk S.F. Liu S. Robins M.J. Borchardt R.T. Biochemistry. 1994; 33: 12305-12311Crossref PubMed Scopus (27) Google Scholar). This assay measures the rate of formation of AdoHcy from Ado and Hcy using HPLC. AdoHcy hydrolase (100 μg) was incubated with or without the photoaffinity probe 8-N3-Ado (50 μM) in 200 μl of 50 mM sodium phosphate buffer, pH 7.2, (buffer A) for 1 h at 37°C. To the reaction mixture was added 3 volumes of 97% ethanol to denature the enzyme. The precipitate was removed by centrifugation, and the supernatant was lyophilized. The residue was then dissolved in 100 μl of water for HPLC analysis using a reverse phase column (Econosphere, C18, 5 μm, 250 × 4.6 mm, Alltech, Deerfield, IL). NAD+ and NADH were eluted from the column isocratically with 0.1 M sodium phosphate buffer, pH 7.0, containing 2.5% of methanol at a flow rate of 1.0 ml/min. Standard curves of NAD+ and NADH were constructed by using known concentrations of freshly prepared authentic NAD+ and NADH. The peaks were monitored at 260 nm. AdoHcy hydrolase (23.5 μg) was incubated with [2-3H]8-N3-Ado (50 μM, 50 μCi/μmol) in the presence or absence of Ado in 50 μl of buffer A at 37°C for 1 h. The reaction mixture was chilled to 0°C and loaded onto a piece of glass plate, which was placed on an ice bath. The photolysis was performed by irradiating the sample with a minelight hand lamp (UVGL-25, 1400 μW/cm2) at 254 nm from a height of 7.6 cm for 2 min. Total binding was determined by a gel filtration method using a Sephadex G-50 spin column. The column (3 ml) equilibrated with buffer A was prespun at 400 × g for 5 min using a swing rotor. The photolyzed protein sample was diluted to 300 μl with buffer A and loaded onto the prespun column. The column was then spun for another 5 min at the same speed. The eluate was collected in a test tube which holds the spin column during centrifugation. The protein concentration of the eluate was determined by the Bradford method and the radioactivity was determined by liquid scintillation counting. The amount of covalent binding was determined by denaturing the protein before gel filtration. The photolyzed sample was mixed with 250 μl of 10 M urea containing 10 mM dithiothreitol, and heated at 100°C for 3 min. After cooling to room temperature, the sample was loaded into a prespun Sephadex G-50 column equilibrated with 1 M urea in buffer A. The column was then spun for 5 min at the same speed as that for the prespin. The protein concentration and radioactivity in the eluate were determined as described above. Control experiments were performed under the denatured conditions with the probe alone or with a non-irradiated enzyme-probe mixture. In both cases, there was no detectable radioactivity in the eluate. AdoHcy hydrolase (2 mg) was incubated with [2-3H]8-N3-Ado (50 μCi/μmol) in a protein-to-probe molar ratio of 1:5 at 37°C for 1.5 h. After photolysis at 0°C for 2 min, the photolabeled protein was filtered through a Sephadex G-50 spin column equilibrated with 100 mM NH4HCO3, pH 8.4, (buffer B) (a 3-ml column for each 300 μl of protein sample) as described above. The eluted protein was denatured by adding solid urea to a final concentration of 8 M. The denatured protein was dialyzed against buffer B containing 2 M urea for 16 h with 2 buffer changes followed by another 6 h of dialysis against buffer B without urea. The dialyzed protein was digested with trypsin in an enzyme-to-substrate ratio (w/w) of 1:20 at 37°C for 14 h. This was followed by addition of another 5% of trypsin for further digestion for 4 h. The boronate affinity chromatography was performed essentially as described by Haley et al.(30Chavan A.J. Nemoto Y. Narumiya S. Kozaki S. Haley B.E. J. Biol. Chem. 1992; 267: 14866-14870Abstract Full Text PDF PubMed Google Scholar). The digested protein was diluted with 100 mM ammonium acetate, pH 8.9 (buffer C), and loaded onto an Affi-Gel 601 column (3 ml) equilibrated with buffer C. The column was washed with 20 ml of buffer C, 10 ml of buffer C containing 0.5 M NaCl, 10 ml of buffer C containing 4 M urea, 20 ml of buffer C containing 100 mM sorbitol, and finally 10 ml of 0.1 M acetic acid. Fractions of 1 ml were collected. Radioactivity in each fraction was determined by liquid scintillation counting, and peptide concentration was monitored at 220 nm with appropriate elution solutions as references. The non-absorbed radioactive fractions were loaded onto another Affi-Gel 601 column to ensure that the radioactive peptides in these fractions were not due to overloading of the column. The pooled, non-absorbed radioactive fractions were then concentrated by lyophilization. The photolabeled peptides collected from the non-absorbed radioactive fractions from the boronate affinity column were purified by reverse phase HPLC on a Vydac C18 Protein and Peptide column (Vydac 218 TP54, C18, 5 μ, 250 × 4.6 mm). The solvent system consisted of mobile phase I (0.1% trifluoroacetic acid) and mobile phase II (80% CH3CN/20% H2O/0.07% trifluoroacetic acid). The elution was carried out with 2% of mobile phase II in mobile phase I with a linear gradient to 70% mobile phase II over 120 min at a flow rate of 0.5 ml/min. The UV absorbance of the eluted peptides was monitored at 220 nm. The radioactivity in the fractions collected (0.5 ml) was measured by liquid scintillation counting. Peptide peaks containing major radioactivity were collected, concentrated by speed vacuum, and rechromatographed on the same column using elution conditions of 20% of mobile phase II with a linear gradient to 60% mobile phase II over 60 min. Peaks were collected manually and radioactivity was determined. Radioactive peaks were rechromatographed again with the same elution conditions as described above. The manually collected peptide peaks containing radioactivity significantly above background were sequenced by automated Edman degradation on an Applied Biosystem 473A Protein Sequencer at the Kansas State University Biotechnology Laboratory, Manhattan, KS. At each sequencing cycle, the washing from the conversion flask and eluate from the HPLC were collected for determination of radioactivity. The photoaffinity probe 8-N3-Ado was used previously to study the relationship between the Ado and cyclic AMP-binding sites on AdoHcy hydrolase purified from human placenta(25Aiyar V.N. Hershfield M.S. Biochem. J. 1985; 232: 643-650Crossref PubMed Scopus (11) Google Scholar). It was shown that 8-N3-Ado was a substrate for the the first step (oxidation of the 3′-hydroxyl group) in the catalytic mechanism of AdoHcy hydrolase, resulting in conversion of E·NAD+ to E·NADH and formation of 3′-keto-8-N3-Ado bound to the active site of the enzyme. Based on this information, we felt that advantage could be taken of the substrate activity of 8-N3-Ado to differentiate specifically photolabeled peptides from nonspecifically photolabeled peptides, a problem that often causes difficulties in photoaffinity labeling experiments. Since the earlier work by Hershfield et al. (25Aiyar V.N. Hershfield M.S. Biochem. J. 1985; 232: 643-650Crossref PubMed Scopus (11) Google Scholar) was done using AdoHcy hydrolase isolated from human placenta while our studies were done with the recombinant human placental enzyme, we first conducted experiments to confirm the specificity of photolabeling of AdoHcy hydrolase with 8-N3-Ado. To show the specificity of the photaffinity probe, the photolabeling should be saturable and the photolabeling protected by a natural substrate of the enzyme. As shown in Fig. 1, when the recombinant human placental AdoHcy hydrolase was photolabeled with increasing concentrations of [2-3H]8-N3-Ado, the extent of labeling approached saturation at approximately 40 μM. When the enzyme was incubated with 50 μM [2-3H]8-N3-Ado in the presence of increasing concentrations of Ado, a natural substrate of the enzyme, but without irradiation, 95% of the total binding of the photolabeling probe was inhibited at 200 μM Ado (Fig. 2). This result indicated that 8-N3-Ado and Ado bind to the same site on AdoHcy hydrolase. An apparent dissociation constant (Kd) of 6.8 μM for 8-N3-Ado was calculated using a computer-aided curve fitting with the following equation: PAAdo/PA = (Kd+[A])/[Kd(1+[Ado]/K'd)+[A]]×100(Eq. 1) where PAAdo was the protein-photolabeling probe complex formed in the presence of Ado; PA was the complex formed in the absence of Ado; A was the concentration of 8-N3-Ado; and Kd′ was the dissociation constant (4.5 μM) of Ado determined separately by equilibrium dialysis. However, upon irradiation of the enzyme with [2-3H]8-N3-Ado in the presence of Ado, the protection was only 60% at 200 μM Ado, and further increases of Ado concentration did not afford better protection (Fig. 2). This suggested that not all photolabeling occurred in the Ado-binding site and that some of the 8-N3-Ado must have nonspecifically photolabeled the protein outside of the Ado-binding site, which could not be protected by Ado. summarizes the results of binding, photoincorporation, and inactivation of AdoHcy hydrolase by 8-N3-Ado. Incorporation of [2-3H]8-N3-Ado to the protein was light-dependent. Incubation of the enzyme with the photolabeling probe in the absence of light gave a binding stoichiometery of 0.81 ± 0.05 mol of probe/mol enzyme subunit, but without covalent incorporation. After irradiation, the total binding increased to 0.95 ± 0.06 mol of probe/mol of enzyme subunit, in which 0.29 ± 0.05 mol of the probe was covalently photoinserted into the protein. However, about 40% of the covalent modification could not be protected by high concentrations of Ado, indicating the existence of significant nonspecific photoinsertion. This result was in agreement with the earlier observation that 20-60% of the covalent labeling of AdoHcy hydrolase with 8-N3-Ado could not be blocked by Ado (25Aiyar V.N. Hershfield M.S. Biochem. J. 1985; 232: 643-650Crossref PubMed Scopus (11) Google Scholar). Subtracting the nonspecific labeling from the total photoinsertion resulted in a net amount of specific photoincorporation of 18%, which is comparable to the reported value of 5-14%(25Aiyar V.N. Hershfield M.S. Biochem. J. 1985; 232: 643-650Crossref PubMed Scopus (11) Google Scholar). The photoincorporation and specificity varied with the enzyme probe ratio. An enzyme/probe ratio of 1:4-6 was found to give the highest photoincorporation with the lowest nonspecific labeling under the conditions of 2 min of irradiation. Irradiation caused a time-dependent loss of the enzyme activity. About 15% of the enzyme activity was lost in the 2-min irradiation in the absence of the probe, and 56% was lost in the presence of the probe. This increased loss of the enzyme activity was prevented by inclusion of 200 μM Ado (Table I). 8-N3-Ado in the absence of irradiation was a slow time-dependent inhibitor of AdoHcy hydrolase. Incubation of the enzyme with 50 μM of 8-N3-Ado at 37°C for 1 h in the absence of light resulted in 51% inactivation of the enzyme. This enzyme inactivation could be protected by Ado, supporting the idea that the probe was interacting at the active site of the enzyme.Figure 2:Effect of Ado on binding and photoinsertion of [2-3H]8-N3-Ado into AdoHcy hydrolase. Recombinant human placental AdoHcy hydrolase (23.5 μg) was incubated with 50 μM of [2-3H]8-N3-Ado (50 μCi/μmol) in the absence and presence of increasing concentrations of Ado in 50 μl of buffer A at 37°C for 1 h. Non-irradiated samples (○) were used to determine the total binding, and irradiated samples (●) were used to determine the covalent binding as described under “Experimental Procedures.” The ratio of the binding stoichiometry (PA/protein-photolabeling probe complex) in the presence of Ado to that in the absence of Ado was plotted against Ado concentration.View Large Image Figure ViewerDownload Hi-res image Download (PPT)TABLE IBinding, photoincorporation, and inactivation of AdoHcy hydrolase by 8-N3-Ado Open table in a new tab Because of the high percentage of nonspecific photoinsertion, one might, at first glance, question the strategy of identifying specifically photolabeled peptides of AdoHcy hydrolase using 8-N3-Ado as a probe. However, if one takes advantage of 8-N3-Ado being a substrate of the oxidative activity of the enzyme(25Aiyar V.N. Hershfield M.S. Biochem. J. 1985; 232: 643-650Crossref PubMed Scopus (11) Google Scholar), it immediately becomes apparent that it should be possible to separate the peptides specifically labeled with 3′-keto-8-N3-Ado from those nonspecifically labeled by 8-N3-Ado by using boronate affinity chromatography. Boronate resin has a strong affinity for compounds that have adjacent cis-hydroxyl group(s) (cis-diols) and has thus been used to separate deoxyribonucleotides from ribonucleotides effectively(31Schott H. Rudloff E. Schmidt P. Roychoudhury R. Kosscl H. Biochemistry. 1973; 12: 932-938Crossref PubMed Scopus (101) Google Scholar). 8-N3-Ado bound at the enzyme active site will participate in the enzyme-catalyzed oxidation reaction, which reduces E·NAD+ to E·NADH with the concomitant formation of 3′-keto-8-N3-Ado(25Aiyar V.N. Hershfield M.S. Biochem. J. 1985; 232: 643-650Crossref PubMed Scopus (11) Google Scholar). The NADH form of the enzyme, in turn, tightly binds the 3′-keto product in its active site(32Yuan C.S. Yeh J. Squier T.C. Rawitch A. Borchardt R.T. Biochemistry. 1993; 32: 10414-10422Crossref PubMed Scopus (16) Google Scholar), which becomes covalently bound after irradiation. Peptides labeled with 3′-keto probe are thus assumed to be parts of the adenine (Ade)-binding domain. Because the 3′-keto probe lacks adjacent cis-hydroxyl groups, peptides labeled by 3′-keto-8-N3-Ado should have no affinity to the boronate resin and should be eluted from the column directly without retardation (non-absorbed fractions). In contrast, peptides nonspecifically modified by 8-N3-Ado should possess adjacent cis-hydroxyl groups (2′ and 3′- hydroxyl group) and hence have a strong affinity to the boronate resin and remain in the column. In order to utilize this affinity separation technique, we first undertook an experiment to confirm that 8-N3-Ado serves as a substrate for the oxidative activity of the enzyme by monitoring the conversion of E·NAD+ to E·NADH after incubation of the enzyme with the probe. As shown in Fig. 3, the purified AdoHcy hydrolase contains about 0.85 mol of NAD+ and 0.1 mol of NADH/mol of enzyme subunit (Fig. 3a). After incubation with 50 μM of 8-N3-Ado at 37°C for 1 h, about 53% of the NAD+ was converted to NADH (Fig. 3b). Prolonged incubation did not significantly increase the ratio of NAD+/NADH. This result confirmed the earlier observation that 8-N3-Ado participates as a substrate in the enzyme-catalyzed oxidation reaction. After irradiation of the reaction mixture of AdoHcy hydrolase and 8-N3-Ado, the protein was denatured by treatment with 8 M urea and subsequent heating. The free probe was removed by gel filtration and dialysis. The labeled protein was digested with trypsin, and the resulting peptides were subjected to boronate affinity chromatography. Fig. 4a shows a typical elution profile of the photolabeled tryptic peptides on boronate affinity chromatography. Two major radioactive peaks were observed; one eluted with the non-absorbed fractions (fractions 2-7) and accounted for about 40% of the total radioactivity, and the second was eluted by 100 mM sorbitol (fractions 44-56). To confirm that the radioactive peptides in the non-absorbed fractions were not due to overloading the column, those fractions were pooled and applied to another boronate column. From this chromatography, more than 95% of the radioactivity was recovered from the non-absorbed fractions (data not shown). This result indicates that the non-absorbed radioactive peptides contain no adjacent cis-hydroxyl group(s) and were more than likely labeled by 3′-keto-8-N3-Ado. The percent (40%) of specifically photolabeled peptides from the affinity column was lower than that (60%) observed from the Ado protection experiment, indicating that some of the active site peptides were labeled by 8-N3-Ado rather than its 3′-keto derivative. This was expected since incubation of the enzyme with 8-N3-Ado at 37°C for 1 h could convert only 53% of the E·NAD+ to E·NADH, and the rest of the probe bound in the active site still existed in its original non-oxidized form at the time point of irradiation. Nevertheless, this part of the specifically labeled peptides was sacrificed in the separation method. To further confirm the specificity of the photoinsertion by 8-N3-Ado, a parallel photolabeling experiment was carried out using apoAdoHcy hydrolase which lacks NAD+ and thus does not catalyze 3′-oxidation of substrates. However, apoAdoHcy hydrolase still binds Ado and catalyzes the 5′-hydrolytic reaction (28Yuan C.S. Yeh J. Liu S. Borchardt R.T. J. Biol. Chem. 1993; 268: 17030-17037Abstract Full Text PDF PubMed Google Scholar). As shown in Fig. 4b, approximately 95% of the total radioactivity from the 8-N3-Ado-apoAdoHcy hydrolase experiment was retained on the boronate affinity resin and was eluted specifically by sorbitol. The small amount (5%) of the radioactivity that elutes with the non-absorbed fractions most likely arises from the residual NAD+ form of the enzyme present in the apo enzyme. Comparison of the elution profiles of boronate affinity chromatographies of the NAD+ form and the apo form of the enzyme photolabeled with [3H]8-N3-Ado gives strong evidence that radioactive peptides in the non-absorbed fractions from the affinity column are those specifically photolabeled by enzymatically oxidized 8-N3-Ado, presumably 3′-keto-8-N3-Ado. When the non-absorbed radioactive fractions in Fig. 4a were pooled and analyzed by reverse phase HPLC, the chromatograph showed two major radioactive peaks: peak a (fraction number 40-43) and peak b (fraction number 44-46) (Fig. 5). These two peaks account for about 70% of the total radioactivity. These two peaks were pooled separately and rechromatographed by reverse phase HPLC by manually collecting eluted peaks. Peaks associated with significant radioactivity were rechromatographed. As shown in Fig. 6, rechromatography generated a major and several minor components with the radioactivity associated only with the major components, peak a′ and peak b′, which were isolated from peaks a and b, respectively.Figure 6:Rechromatography of photolabeled peptides purified from HPLC. Peak a (fraction 40-43) and peak b (fraction 44-46) in Fig. 5 were pooled, concentrated, and loaded onto a reverse phase HPLC column (Vydac 218TP54, C18, 5 μ, 250 × 4.6 mm). Peptides were eluted by a gradient of mobile phase II (acetonitrile/H2O, 0.07%trifluoroacetic acid) in mobile phase I (0.1% trifluoroacetic acid) from 20 to 60% over 60 min at a flow rate of 0.5 ml/min. Peaks were collected manually, and radioactivity was determined for each peak. The major radioactive peaks were loaded onto the same column again and eluted under the same conditions. Shadowed peaks were radioactive and were manually collected for amino acid sequencing.View Large Image Figure ViewerDownload Hi-res image Download (PPT) Upon amino acid sequence analysis, peak a′ was found to contain peptide Val319-Arg327 (peptide I), and peak b′ contained peptide Val175-Lys186 (peptide II) as seen in Table II. Liquid scintillation counting of the washing from the conversion flask and the eluate from HPLC at each cycle of sequencing showed that Ile321 in peptide I and Ala177 in peptide II were associated with the highest radioactivity in the two peptides. These results indicate that Ile321 and Ala177 are the photoinsertion sites. Since both Ile321 and Ala177 were eluted with normal retention times from the reverse phase HPLC during sequencing, it is likely that the 3H label dissociated from the modified amino acid residue during the sequencing. In a separate experiment, the photolabeled peptides were found to be acid-labile. Upon exposure of the labeled peptides to anhydrous acetic acid, the 3H label completely dissociated from peptides. Therefore, it is probable that during the sequencing, the 3H label dissociated in the conversion flask where anilinothiozolinone amino acids are converted to phenythiohydantoin derivatives at high temperature (~64°C) and low pH (<2.0).TABLE IIAmino acid sequence analysis of the photolabeled peptides Open table in a new tab Comparison of amino acid sequences of AdoHcy hydrolase from different sources has provided useful information about the structure-function relationships of the enzyme and the identity of essential amino acid residues involved in enzyme catalysis or substrate/cofactor binding(5Creedon K.A. Rathod P.K. Wellems T.E. J. Biol. Chem. 1994; 269: 16364-16370Abstract Full Text PDF PubMed Google Scholar, 21Aksamit R.R. Backlund Jr., P.S. Moos Jr., M. Caryk T. Gomi T. Ogawa H. Fujioka M. Cantoni G.L. J. Biol. Chem. 1994; 269: 4084-4091Abstract Full Text PDF PubMed Google Scholar). AdoHcy hydrolase has been cloned from a range of sources representing billions of years of evolution. The alignment of entire sequences of AdoHcy hydrolase from different sources has been published (5Creedon K.A. Rathod P.K. Wellems T.E. J. Biol. Chem. 1994; 269: 16364-16370Abstract Full Text PDF PubMed Google Scholar). Fig. 7 shows only the sequences in the two photolabeled regions. Both of the photolabeled peptides are located in very conserved regions of the entire gene. For example, with the exception of the first residue, Val175, the photolabeled region of peptide II is almost completely conserved in all known AdoHcy hydrolases. The only exceptions are the P. falciparum enzyme, where 3 residues are replaced, and the L. donovani enzyme, where 2 residues are substituted. Nevertheless, the photoinsertion site Ala177 is conserved in all known AdoHcy hydrolases. Interestingly, peptide II is located very close to Cys195, a residue that was identified as being located in the active site of the enzyme by chemical modification.2( 2C. S. Yuan, D. B. Ault-Riche, and R. T. Borchardt, manuscript in preparation. )Moreover, limited proteolysis studies on human AdoHcy hydrolase from our laboratory have recently demonstrated that Glu197-Ser198 is located in or near the active site of the enzyme by showing that cleavage of the peptide bond between Glu197-Ser198 by protease Staphylococcus aureus strain V8 on a mutant form (K426E) of the enzyme was specifically protected by the substrate Ado(22Gupta R.A. Yuan C.S. Ault-Riche D.B. Borchardt R.T. Arch. Biochem. Biophys. 1995; 319: 365-371Crossref PubMed Scopus (6) Google Scholar). Information from these two separate studies gives further support to the idea that peptide II could be located in or near the active or substrate-binding site of the enzyme. In fact, Ala177 may be located very close to the C-8 position of Ade in the three-dimensional structure of the enzyme, at least after conformational changes induced by the probe (32Yuan C.S. Yeh J. Squier T.C. Rawitch A. Borchardt R.T. Biochemistry. 1993; 32: 10414-10422Crossref PubMed Scopus (16) Google Scholar). For the photolabeled peptide I (Val319-Arg327), a region from the third residue (Ile321) of the peptide to Arg327 is well conserved with only a few substitutions observed in enzymes from lower evolved cells such as parasites and bacteria. In fact, this region was predicted to be involved in Ade ring binding by a computer graphics model developed in our laboratory, which shows that in the Ade portion of substrates and inhibitors, the C-6-NH2 group interacts with the main-chain carbonyl group of Leu329 (Leu330 in human) for recombinant rat liver enzyme(33Yeh J.C. Borchardt R.T. Vedani A. J. Comput. Aided Mol. Design. 1991; 5: 213-234Crossref PubMed Scopus (14) Google Scholar). Since Leu330 in the human enzyme is only 9 residues away from the photoinsertion site Ile321, it is likely that Ile321 is positioned such that it is close enough to the C-8 of the Ade ring to which (C-8) the azido group is attached in the photoaffinity probe. Therefore, based on results from this and earlier studies, we believe that the two photolabeled peptides Val175-Lys186 and Val319-Arg327 could be parts of the Ade ring-binding domain in the substrate binding site of AdoHcy hydrolase. AdoHcy hydrolase (23.5 μg) was incubated with 50 μM of [2-3H]8-N3-Ado in 50 μl of buffer A at 37°C for 1 h in the presence and absence of Ado followed by irradiation at 0°C for 2 min. The remaining enzyme activity was determined in the synthetic direction as described under “Experimental Procedures.” Stoichiometries of the total binding were determined by gel filtration under non-denatured conditions, and the covalent binding was determined by treating samples with 8 M urea containing 10 mM dithiothreitol and heating at 100°C for 3 min before gel filtration under denatured conditions as described under “Experimental Procedures.” We acknowledge Dr. Michael Hershfield for providing us with a sample of E. coli transformed with a plasmid encoding for human placental AdoHcy hydrolase." @default.
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