FRINK Linked Data Fragments server

Matches in SemOpenAlex for { <https://semopenalex.org/work/W2080510513> ?p ?o ?g. }

• W2080510513 endingPage "9732" @default.
• W2080510513 startingPage "9722" @default.
• W2080510513 abstract "Thimet oligopeptidase (EC 3.4.24.15) and neurolysin (EC 3.4.24.16) are closely related zinc-dependent metallopeptidases that metabolize small bioactive peptides. They cleave many substrates at the same sites, but they recognize different positions on others, including neurotensin, a 13-residue peptide involved in modulation of dopaminergic circuits, pain perception, and thermoregulation. On the basis of crystal structures and previous mapping studies, four sites (Glu-469/Arg-470, Met-490/Arg-491, His-495/Asn-496, and Arg-498/Thr-499; thimet oligopeptidase residues listed first) in their substrate-binding channels appear positioned to account for differences in specificity. Thimet oligopeptidase mutated so that neurolysin residues are at all four positions cleaves neurotensin at the neurolysin site, and the reverse mutations in neurolysin switch hydrolysis to the thimet oligopeptidase site. Using a series of constructs mutated at just three of the sites, it was determined that mutations at only two (Glu-469/Arg-470 and Arg-498/Thr-499) are required to swap specificity, a result that was confirmed by testing the two-mutant constructs. If only either one of the two sites is mutated in thimet oligopeptidase, then the enzyme cleaves almost equally at the two hydrolysis positions. Crystal structures of both two-mutant constructs show that the mutations do not perturb local structure, but side chain conformations at the Arg-498/Thr-499 position differ from those of the mimicked enzyme. A model for differential recognition of neurotensin based on differences in surface charge distribution in the substrate binding sites is proposed. The model is supported by the finding that reducing the positive charge on the peptide results in cleavage at both hydrolysis sites. Thimet oligopeptidase (EC 3.4.24.15) and neurolysin (EC 3.4.24.16) are closely related zinc-dependent metallopeptidases that metabolize small bioactive peptides. They cleave many substrates at the same sites, but they recognize different positions on others, including neurotensin, a 13-residue peptide involved in modulation of dopaminergic circuits, pain perception, and thermoregulation. On the basis of crystal structures and previous mapping studies, four sites (Glu-469/Arg-470, Met-490/Arg-491, His-495/Asn-496, and Arg-498/Thr-499; thimet oligopeptidase residues listed first) in their substrate-binding channels appear positioned to account for differences in specificity. Thimet oligopeptidase mutated so that neurolysin residues are at all four positions cleaves neurotensin at the neurolysin site, and the reverse mutations in neurolysin switch hydrolysis to the thimet oligopeptidase site. Using a series of constructs mutated at just three of the sites, it was determined that mutations at only two (Glu-469/Arg-470 and Arg-498/Thr-499) are required to swap specificity, a result that was confirmed by testing the two-mutant constructs. If only either one of the two sites is mutated in thimet oligopeptidase, then the enzyme cleaves almost equally at the two hydrolysis positions. Crystal structures of both two-mutant constructs show that the mutations do not perturb local structure, but side chain conformations at the Arg-498/Thr-499 position differ from those of the mimicked enzyme. A model for differential recognition of neurotensin based on differences in surface charge distribution in the substrate binding sites is proposed. The model is supported by the finding that reducing the positive charge on the peptide results in cleavage at both hydrolysis sites. Bioactive peptides that serve as signaling molecules in the central nervous system and periphery are inactivated or modified by a group of enzymes known as neuropeptidases. With few exceptions, these enzymes are metallopeptidases that carry out peptide bond hydrolysis with the assistance of a zinc ion cofactor. The neuropeptidases thimet oligopeptidase (TOP, 3The abbreviations used are: TOP, thimet oligopeptidase; NT, neurotensin; NT(R9E), neurotensin modified peptide (Arg-9 → Glu); TOP4, thimet oligopeptidase mutated to the neurolysin sequence at four residue positions; neurolysin 4, neurolysin mutated to the thimet oligopeptidase sequence at four residue positions; TOP2, thimet oligopeptidase mutated to the neurolysin sequence at two residue positions; neurolysin 2, neurolysin mutated to the thimet oligopeptidase sequence at two residue positions; r.m.s., root mean square; HPLC, high pressure liquid chromatography. 3The abbreviations used are: TOP, thimet oligopeptidase; NT, neurotensin; NT(R9E), neurotensin modified peptide (Arg-9 → Glu); TOP4, thimet oligopeptidase mutated to the neurolysin sequence at four residue positions; neurolysin 4, neurolysin mutated to the thimet oligopeptidase sequence at four residue positions; TOP2, thimet oligopeptidase mutated to the neurolysin sequence at two residue positions; neurolysin 2, neurolysin mutated to the thimet oligopeptidase sequence at two residue positions; r.m.s., root mean square; HPLC, high pressure liquid chromatography. EC 3.4.24.15) and neurolysin (EC 3.4.24.16) are closely related members of the zinc metallopeptidase M3 family (1Barrett A.J. Brown M.A. Dando P.M. Knight C.G. McKie N. Rawlings N.D. Serizawa A. Methods Enzymol. 1995; 248: 529-556Crossref PubMed Scopus (86) Google Scholar, 2Rawlings N.D. Barrett A.J. Methods Enzymol. 1995; 248: 183-228Crossref PubMed Scopus (688) Google Scholar, 3Orlowski M. Michaud C. Chu T.G. Eur. J. Biochem. 1983; 135: 81-88Crossref PubMed Scopus (244) Google Scholar, 4Orlowski M. Reznik S. Ayala J. Pierotti A.R. Biochem. J. 1989; 261: 951-958Crossref PubMed Scopus (99) Google Scholar, 5Pierotti A. Dong K.W. Glucksman M.J. Orlowski M. Roberts J.L. Biochemistry. 1990; 29: 10323-10329Crossref PubMed Scopus (119) Google Scholar, 6Checler F. Vincent J.P. Kitabgi P. J. Biol. Chem. 1986; 261: 11274-11281Abstract Full Text PDF PubMed Google Scholar, 7Dauch P. Vincent J.P. Checler F. J. Biol. Chem. 1995; 270: 27266-27271Abstract Full Text Full Text PDF PubMed Scopus (61) Google Scholar, 8Wilk S. Orlowski M. J. Neurochem. 1980; 35: 1172-1182Crossref PubMed Scopus (251) Google Scholar, 9Wilk S. Pearce S. Orlowski M. Life Sci. 1979; 24: 457-464Crossref PubMed Scopus (32) Google Scholar). They contain a thermolysin-like catalytic domain that includes a His-Glu-Xaa-Xaa-His (HEXXH) sequence motif involved in zinc ion coordination and catalysis. Both enzymes are widely distributed in mammalian tissues and are found in different subcellular locations (5Pierotti A. Dong K.W. Glucksman M.J. Orlowski M. Roberts J.L. Biochemistry. 1990; 29: 10323-10329Crossref PubMed Scopus (119) Google Scholar, 10Checler F. Barelli H. Dauch P. Dive V. Vincent B. Vincent J.P. Methods Enzymol. 1995; 248: 593-614Crossref PubMed Scopus (33) Google Scholar, 11Acker G.R. Molineaux C. Orlowski M. J. Neurochem. 1987; 48: 284-292Crossref PubMed Scopus (118) Google Scholar, 12Chu T.G. Orlowski M. Endocrinology. 1985; 116: 1418-1425Crossref PubMed Scopus (141) Google Scholar, 13Healy D.P. Orlowski M. Brain Res. 1992; 571: 121-128Crossref PubMed Scopus (57) Google Scholar, 14Vincent B. Beaudet A. Dauch P. Vincent J.-P. Checler F. J. Neurosci. 1996; 16: 5049-5059Crossref PubMed Google Scholar, 15Kato A. Sugiura N. Saruta Y. Hosoiri T. Yasue H. Hirose S. J. Biol. Chem. 1997; 272: 15313-15322Abstract Full Text Full Text PDF PubMed Scopus (41) Google Scholar, 16Crack P.J. Wu T.J. Cummins P.M. Ferro E.S. Tullai J.W. Glucksman M.J. Roberts J.L. Brain Res. 1999; 835: 113-124Crossref PubMed Scopus (59) Google Scholar, 17Jeske N.A. Glucksman M.J. Roberts J.L. J. Neurosci. Res. 2003; 74: 468-473Crossref PubMed Scopus (16) Google Scholar, 18Jeske N.A. Glucksman M.J. Roberts J.L. J. Neurochem. 2004; 90: 819-828Crossref PubMed Scopus (31) Google Scholar, 19Dando P.M. Brown M.A. Barrett A.J. Biochem. J. 1993; 294: 451-457Crossref PubMed Scopus (55) Google Scholar, 20Ferro E.S. Tullai J.W. Glucksman M.J. Roberts J.L. DNA Cell Biol. 1999; 18: 781-789Crossref PubMed Scopus (51) Google Scholar, 21Ferro E.S. Carreno F.R. Goni C. Garrido P.A. Guimaraes A.O. Castro L.M. Oliveira V. Araujo M.C. Rioli V. Gomes M.D. Fontenele-Neto J.D. Hyslop S. Protein Pept. Lett. 2004; 11: 415-421Crossref PubMed Scopus (19) Google Scholar), where they primarily metabolize small, bioactive peptides involved in a range of physiological processes. In addition, TOP has been shown to degrade peptides released by the proteasome, limiting the extent of antigen presentation by major histocompatibility complex class I molecules (22Kim S.I. Pabon A. Swanson T.A. Glucksman M.J. Biochem. J. 2003; 375: 111-120Crossref PubMed Scopus (38) Google Scholar, 23Portaro F.C. Gomes M.D. Cabrera A. Fernandes B.L. Silva C.L. Ferro E.S. Juliano L. de Camargo A.C. Biochem. Biophys. Res. Commun. 1999; 255: 596-601Crossref PubMed Scopus (49) Google Scholar, 24Saric T. Beninga J. Graef C.I. Akopian T.N. Rock K.L. Goldberg A.L. J. Biol. Chem. 2001; 276: 36474-36481Abstract Full Text Full Text PDF PubMed Scopus (123) Google Scholar, 25Saric T. Graef C.I. Goldberg A.L. J. Biol. Chem. 2004; 279: 46723-46732Abstract Full Text Full Text PDF PubMed Scopus (152) Google Scholar, 26Silva C.L. Portaro F.C. Bonato V.L. de Camargo A.C. Ferro E.S. Biochem. Biophys. Res. Commun. 1999; 255: 591-595Crossref PubMed Scopus (70) Google Scholar, 27York I.A. Mo A.X. Lemerise K. Zeng W. Shen Y. Abraham C.R. Saric T. Goldberg A.L. Rock K.L. Immunity. 2003; 18: 429-440Abstract Full Text Full Text PDF PubMed Scopus (129) Google Scholar), and it has been associated with amyloid protein precursor processing (28Yamin R. Malgeri E.G. Sloane J.A. McGraw W.T. Abraham C.R. J. Biol. Chem. 1999; 274: 18777-18784Abstract Full Text Full Text PDF PubMed Scopus (88) Google Scholar). Human neurolysin and TOP share 63% sequence identity over 677 common residues, and the crystal structures of both enzymes (29Brown C.K. Madauss K. Lian W. Beck M.R. Tolbert W.D. Rodgers D.W. Proc. Natl. Acad. Sci. U. S. A. 2001; 98: 3127-3132Crossref PubMed Scopus (120) Google Scholar, 30Ray K. Hines C.S. Coll-Rodriguez J. Rodgers D.W. J. Biol. Chem. 2004; 279: 20480-20489Abstract Full Text Full Text PDF PubMed Scopus (75) Google Scholar) show that they adopt almost identical folds, with an r.m.s. deviation on Cα atoms of only 1.2 Å. Each enzyme is split into two domains by a deep, narrow channel that runs the length of the molecule, and the active site is located near the bottom of this groove. The neurolysin/TOP fold is conserved in the M3 family member dipeptidyl carboxypeptidase (31Comellas-Bigler M. Lang R. Bode W. Maskos K. J. Mol. Biol. 2005; 349: 99-112Crossref PubMed Scopus (37) Google Scholar), as well as the more distantly related metallopeptidases angiotensin-converting enzyme (32Natesh R. Schwager S.L. Sturrock E.D. Acharya K.R. Nature. 2003; 421: 551-554Crossref PubMed Scopus (665) Google Scholar), angiotensin converting enzyme-related carboxypeptidase (33Towler P. Staker B. Prasad S.G. Menon S. Tang J. Parsons T. Ryan D. Fisher M. Williams D. Dales N.A. Patane M.A. Pantoliano M.W. J. Biol. Chem. 2004; 279: 17996-18007Abstract Full Text Full Text PDF PubMed Scopus (459) Google Scholar), and Pyrococcus furiosus carboxypeptidase (34Arndt J.W. Hao B. Ramakrishnan V. Cheng T. Chan S.I. Chan M.K. Structure (Camb.). 2002; 10: 215-224Abstract Full Text Full Text PDF PubMed Scopus (54) Google Scholar). Shielding of the active site by the channel walls in neurolysin and TOP, as well as the structurally related enzymes, restricts them to small peptide substrates (35Konkoy C.S. Davis T.P. Trends Pharmacol. Sci. 1996; 17: 288-294Abstract Full Text Full Text PDF PubMed Scopus (55) Google Scholar, 36McKelvy J.F. Blumberg S. Annu. Rev. Neurosci. 1986; 9: 415-434Crossref PubMed Scopus (109) Google Scholar, 37Eipper B.A. Mains R.E. Herbert E. Trends Neurosci. 1986; 9: 463-468Abstract Full Text PDF Scopus (73) Google Scholar, 38Littlewood G.M. Iversen L.L. Turner A.J. Neurochem. Int. 1988; 12: 383-389Crossref PubMed Scopus (37) Google Scholar, 39Checler F. Nagatsu T. Parvez H. Naoi M. Parvez S. Methods in Neurotransmitter and Neuropeptide Research. 2. Elsevier, Amsterdam1993: 375-418Google Scholar, 40Csuhai E. Safavi A. Thompson M.W. Hersh L.B. Hook V. Proteolytic and Cellular Mechanisms in Prohormone and Neuropeptide Precursor Processing. Springer-Verlag, Heidelberg1998: 173-189Google Scholar). In addition, these two enzymes share with some other neuropeptidases the ability to recognize highly diverse cleavage site sequences while maintaining specificity for those sites. This fuzzy recognition (41Moodie S.L. Mitchell J.B. Thornton J.M. J. Mol. Biol. 1996; 263: 486-500Crossref PubMed Scopus (110) Google Scholar) allows the enzymes to play different roles in different tissues and subcellular locations, and understanding the mechanism underlying this ability is a key objective in the study of neuropeptidases. Neurolysin and TOP cleave most bioactive peptides at the same site or sites, but they recognize different positions on some naturally occurring and synthetic peptides (1Barrett A.J. Brown M.A. Dando P.M. Knight C.G. McKie N. Rawlings N.D. Serizawa A. Methods Enzymol. 1995; 248: 529-556Crossref PubMed Scopus (86) Google Scholar, 42Dahms P. Mentlein R. Eur. J. Biochem. 1992; 208: 145-154Crossref PubMed Scopus (76) Google Scholar, 43Mentlein R. Dahms P. J. Neurochem. 1994; 64: 27-37Google Scholar, 44Oliveira V. Campos M. Hemerly J.P. Ferro E.S. Camargo A.C. Juliano M.A. Juliano L. Anal. Biochem. 2001; 292: 257-265Crossref PubMed Scopus (36) Google Scholar, 45Oliveira V. Campos M. Melo R.L. Ferro E.S. Carmago A.C.M. Juliano M.A. Julinan L. Biochemistry. 2001; 40: 4417-4425Crossref PubMed Scopus (68) Google Scholar). Notably, they cleave at distinct sites on the 13-residue bioactive peptide neurotensin, for which they are the primary metabolizing enzymes (4Orlowski M. Reznik S. Ayala J. Pierotti A.R. Biochem. J. 1989; 261: 951-958Crossref PubMed Scopus (99) Google Scholar, 6Checler F. Vincent J.P. Kitabgi P. J. Biol. Chem. 1986; 261: 11274-11281Abstract Full Text PDF PubMed Google Scholar, 46Barelli H. Fox-Threlkeld J.E.T. Dive V. Daniel E.E. Vincent J.P. Checler F. Br. J. Pharmacol. 1994; 112: 127-132Crossref PubMed Scopus (39) Google Scholar, 47Chabry J. Checler F. Vincent J.-P. Mazella J. J. Neurosci. 1990; 10: 3916-3921Crossref PubMed Google Scholar, 48Vincent B. Dive V. Yiotakis A. Smadja C. Maldonado R. Vincent J.-P. Checler F. Br. J. Pharmacol. 1995; 115: 1053-1063Crossref PubMed Scopus (33) Google Scholar, 49Barelli H. Vincent J.P. Checler F. Eur. J. Biochem. 1988; 175: 481-489Crossref PubMed Scopus (26) Google Scholar, 50Wilk S. Orlowski M. J. Chromatogr. 1982; 249: 121-129Crossref PubMed Scopus (13) Google Scholar, 51Knight C.G. Dando P.M. Barrett A.J. Biochem. J. 1995; 308: 145-150Crossref PubMed Scopus (26) Google Scholar). Neurotensin (NT) modulates central dopaminergic and cholinergic circuits, and it is associated with thermoregulation, intestinal motility, and antinocioception (52Carraway R. Leeman S.E. J. Biol. Chem. 1973; 248: 6854-6861Abstract Full Text PDF PubMed Google Scholar, 53Goedert M. Trends Neurosci. 1984; 7: 3-5Abstract Full Text PDF Scopus (31) Google Scholar, 54Kasckow J. Nemeroff C.B. Regul. Pept. 1991; 36: 153-164Crossref PubMed Scopus (108) Google Scholar, 55Kinkead B. Nemeroff C.B. Int. Rev. Neurobiol. 2004; 59: 327-349Crossref PubMed Scopus (45) Google Scholar). Neurolysin cleaves the peptide between Pro-10 and Tyr-11, and TOP cleaves between Arg-8 and Arg-9. This difference in site preference represents an opportunity to begin unraveling the basis of substrate recognition in these enzymes. From their crystal structures and mapping studies, four differences in amino acid sequence were identified as potentially accounting for the differences in NT cleavage (30Ray K. Hines C.S. Coll-Rodriguez J. Rodgers D.W. J. Biol. Chem. 2004; 279: 20480-20489Abstract Full Text Full Text PDF PubMed Scopus (75) Google Scholar, 56Ray K. Hines C.S. Rodgers D.W. Protein Sci. 2002; 11: 2237-2246Crossref PubMed Scopus (26) Google Scholar). Here we have mutated these residues in both neurolysin and TOP in order to test their role in mediating differential substrate specificity. We find that by swapping the amino acids at just two of the positions we can swap cleavage sites on the NT peptide, the reengineered neurolysin now cleaving at the wild type TOP site, and vice versa. We discuss these and additional results in light of a possible mechanism for differential recognition. Preparation of TOP and Neurolysin Expression Constructs— Construction of overexpression vectors for human TOP (pET32 vector; Invitrogen) and rat neurolysin (pBAD vector; Invitrogen) has been described (30Ray K. Hines C.S. Coll-Rodriguez J. Rodgers D.W. J. Biol. Chem. 2004; 279: 20480-20489Abstract Full Text Full Text PDF PubMed Scopus (75) Google Scholar, 57Lian W. Chen G. Wu D. Brown C.K. Madauss K. Hersh L.B. Rodgers D.W. Acta Crystallogr. Sect. D. 2000; 56: 1644-1646Crossref PubMed Scopus (190) Google Scholar). Mutagenesis of the coding sequences that produced the substituted proteins was carried out using the QuikChange site-directed mutagenesis kit (Stratagene). Constructs were completely sequenced to confirm the results of the mutagenesis. Expression and Purification—All TOP variants were expressed in Escherichia coli BL21(DE3)RP cells and purified as previously described (30Ray K. Hines C.S. Coll-Rodriguez J. Rodgers D.W. J. Biol. Chem. 2004; 279: 20480-20489Abstract Full Text Full Text PDF PubMed Scopus (75) Google Scholar). Neurolysin variants were overexpressed in E. coli TOP10 cells and purified as reported (57Lian W. Chen G. Wu D. Brown C.K. Madauss K. Hersh L.B. Rodgers D.W. Acta Crystallogr. Sect. D. 2000; 56: 1644-1646Crossref PubMed Scopus (190) Google Scholar). Purified TOP and TOP mutants were dialyzed against 20 mm Tris-HCl (pH 8.0), 5% glycerol, and 5 mm 2-mercaptoethanol and concentrated to 5–10 mg/ml for storage at 4 °C. Purified neurolysin and neurolysin mutants were dialyzed against 20 mm Tris-HCl (pH 7.4) and 100 mm NaCl and concentrated to 10–15 mg/ml for storage at 4 °C. The level of protein purity was assessed by Coomassie-stained SDS-PAGE (supplemental Fig. S1). In all experiments, the wild type and mutant preparations were found to hydrolyze neurotensin substrate at the known TOP and neurolysin cleavage sites (4Orlowski M. Reznik S. Ayala J. Pierotti A.R. Biochem. J. 1989; 261: 951-958Crossref PubMed Scopus (99) Google Scholar, 6Checler F. Vincent J.P. Kitabgi P. J. Biol. Chem. 1986; 261: 11274-11281Abstract Full Text PDF PubMed Google Scholar, 46Barelli H. Fox-Threlkeld J.E.T. Dive V. Daniel E.E. Vincent J.P. Checler F. Br. J. Pharmacol. 1994; 112: 127-132Crossref PubMed Scopus (39) Google Scholar, 47Chabry J. Checler F. Vincent J.-P. Mazella J. J. Neurosci. 1990; 10: 3916-3921Crossref PubMed Google Scholar, 48Vincent B. Dive V. Yiotakis A. Smadja C. Maldonado R. Vincent J.-P. Checler F. Br. J. Pharmacol. 1995; 115: 1053-1063Crossref PubMed Scopus (33) Google Scholar, 49Barelli H. Vincent J.P. Checler F. Eur. J. Biochem. 1988; 175: 481-489Crossref PubMed Scopus (26) Google Scholar, 50Wilk S. Orlowski M. J. Chromatogr. 1982; 249: 121-129Crossref PubMed Scopus (13) Google Scholar, 51Knight C.G. Dando P.M. Barrett A.J. Biochem. J. 1995; 308: 145-150Crossref PubMed Scopus (26) Google Scholar) with only low levels of hydrolysis at other sites. Determination of Primary Cleavage Sites—Cleavage site assays were performed with 600 μm NT or NT(R9E) and 0.5 μm purified proteins in 10 mm HEPES (pH 7.0). NT was obtained from Sigma, and the NT(R9E) was synthesized by Anaspec, Inc. Reactions were incubated at 37 °C for 10 min and then stopped by addition of 0.25% trifluoroacetic acid. The hydrolyzed peptides were separated by reverse phase chromatography using a Waters 4.6 × 150-mm C18 column eluted at a flow rate of 1 ml/min with a linear gradient of 10–50% acetonitrile in 0.1% trifluoroacetic acid. Absorbance was monitored at 214 nm. The products were collected and dried on a centrifugal vacuum evaporator. The fragments' masses were determined by electrospray ionization-time-of-flight mass spectrometry at the Scripps Research Institute Center for Mass Spectrometry. Kinetic Parameters—Analysis of the steady state kinetics for the TOP and neurolysin constructs was performed with 5–6 nm enzyme, 1–6 μm fluorogenic NT peptide. The fluorogenic peptide (Peptides International) was synthesized with an N-terminal fluorescent 2-aminobenzoyl group and a C-terminal quenching ethylenediaminodinitrophenol group. The hydrolysis reactions were carried out in 25 mm HEPES (pH 7.5), 10 mm NaCl, and 2 mm 2-mercaptoethanol. The fluorescence increase on hydrolysis was followed at 420 nm (excitation at 320 nm) in an LS55 luminescence spectrometer (PerkinElmer Life Sciences). The change in fluorescence intensity over time was converted into rate of product formation at each substrate concentration. The conversion factor (200 fluorescence units/μmol of product) was determined by reactions allowed to proceed to completion. The kinetic parameters were calculated by fitting initial velocity versus substrate concentration to the Michaelis-Menten equation, V = Vmax [S]/(Km + [S]), using nonlinear regression (Prism Version 4 software, GraphPad Inc.) (58Gutfreund H. Kinetics for the Life Sciences: Receptors, Transmitters and Catalysts. Cambridge University Press, Cambridge1995Crossref Google Scholar, 59Motulsky H. Christopoulos A. Fitting Models to Biological Data Using Linear and Nonlinear Regression: A Practical Guide to Curve Fitting. Oxford University Press, New York2004Google Scholar). Errors in kcat and Km values were estimated based on the deviation of the observed initial velocities from the model (60Bevington P.R. Robinson D.K. Data Reduction and Error Analysis for the Physical Sciences. 3rd Ed. McGraw-Hill Education, New York2002Google Scholar). High substrate concentrations were not used in determining kinetic parameters, because deviations from ideality were observed. Substrate inhibition effects have been reported in angiotensin-converting enzyme, another peptidase with the TOP/neurolysin fold (61Schullek J.R. Wilson I.B. Peptides. 1989; 10: 431-434Crossref PubMed Scopus (7) Google Scholar). Crystallization of TOP and Neurolysin Mutants—The TOP2 mutant was crystallized by hanging drop vapor diffusion at 4 °C under conditions similar to those described previously (30Ray K. Hines C.S. Coll-Rodriguez J. Rodgers D.W. J. Biol. Chem. 2004; 279: 20480-20489Abstract Full Text Full Text PDF PubMed Scopus (75) Google Scholar). Briefly, 1 μl of 10 mg/ml protein was mixed with 1 μl of well solution containing 100 mm sodium cacodylate (pH 6.5), 100 mm magnesium acetate, 2 mm 2-mercaptoethanol, and 12–14% (w/v) polyethylene glycol 6000. For data collection, crystals were transferred for a few seconds into a solution containing 25% glycerol, 100 mm sodium cacodylate (pH 6.5), 100 mm magnesium acetate, 2 mm 2-mercaptoethanol, and 12–14% (w/v) polyethylene glycol 6000, mounted in a nylon loop (Hampton), and flash-cooled by plunging into liquid nitrogen (62Rodgers D.W. Methods Enzymol. 1997; 276: 183-203Crossref PubMed Scopus (108) Google Scholar). Neurolysin 2 mutant crystals were also obtained by hanging drop vapor diffusion at 4 °C following a procedure similar to that reported (57Lian W. Chen G. Wu D. Brown C.K. Madauss K. Hersh L.B. Rodgers D.W. Acta Crystallogr. Sect. D. 2000; 56: 1644-1646Crossref PubMed Scopus (190) Google Scholar). The well solution for neurolysin was 100 mm sodium cacodylate (pH 6.5), 100 mm magnesium chloride, 0.1 mm zinc chloride, 1 mm 2-mercaptoethanol, and 10–12% (w/v) polyethylene glycol 8000. The crystals were grown by mixing 1–2 μl of 15 mg/ml protein with an equal volume of well solution. In preparation for data collection, crystals were exposed for a few seconds to a solution containing the same components as the well solution plus 20% polyethylene glycol 400 and flash-cooled as described above. Data Collection and Structure Determination—X-ray data were collected at the Advanced Photon Source beamline 22-ID (Southeast Regional Collaborative Access Team), Argonne National Laboratory. Data were reduced with HKL2000 (63Otwinowski Z. Minor W. Methods Enzymol. 1997; 276: 307-326Crossref PubMed Scopus (38445) Google Scholar), and initial structures of TOP2 and neurolysin 2 were determined by molecular replacement with the CNS software package (64Brünger A.T. Adams P.D. Clore G.M. DeLano W.L. Gros P. Grosse-Kunstleve R.W. Jiang J.S. Kuszewski J. Nilges M. Pannu N.S. Read R.J. Rice L.M. Simonson T. Warren G.L. Acta Crystallogr. Sect. D Biol. Crystallogr. 1998; 54: 905-921Crossref PubMed Scopus (16947) Google Scholar) using native TOP and neurolysin coordinates, respectively (Protein Data Bank codes 1S4B and 1I1I). Model building and analysis were performed by using the program O (65Jones T.A. Zou J.Y. Cowan S.W. Kjeldgaard M. Acta Crystallogr. Sect. A. 1991; 47: 110-119Crossref PubMed Scopus (13006) Google Scholar), and structures were refined with CNS. The space group of the TOP2 crystals is P212121, and the cell dimensions are a = 77.1 Å, b = 99.3 Å, c = 105.7 Å. The space group of the neurolysin 2 crystals is P21212 with unit cell dimensions of a = 159.6 Å, b = 87.7 Å, c = 58.4 Å. Residue Positions Possibly Mediating Differential Recognition—Recognition of different hydrolysis sites on the peptide NT by TOP and neurolysin (Fig. 1A) provides an opportunity to begin exploring the basis for their substrate specificity. As we noted in earlier studies (30Ray K. Hines C.S. Coll-Rodriguez J. Rodgers D.W. J. Biol. Chem. 2004; 279: 20480-20489Abstract Full Text Full Text PDF PubMed Scopus (75) Google Scholar, 56Ray K. Hines C.S. Rodgers D.W. Protein Sci. 2002; 11: 2237-2246Crossref PubMed Scopus (26) Google Scholar), only a relatively small number of the sequence differences between the two enzymes map to the interior of the substrate-binding channel, far fewer than expected based on its surface area. Of these sequence differences, the four changes identified as being both conserved in orthologs and well positioned to affect substrate specificity are Glu-469/Arg-470, Met-490/Arg-491, His-495/Asn-496, and Arg-498/Thr-499 (TOP residues listed first; Fig. 1, B and C). Two of the sites (Glu-469/Arg-470 and Arg-498/Thr-499) are relatively close to the active sites of the enzymes, whereas the other two are located near one end of the channel, where they might interact with N-terminal residues of the NT substrate. We suggested (30Ray K. Hines C.S. Coll-Rodriguez J. Rodgers D.W. J. Biol. Chem. 2004; 279: 20480-20489Abstract Full Text Full Text PDF PubMed Scopus (75) Google Scholar) that in the case of neurolysin, Arg-491, Asn-496, and Thr-499 from the enzyme might interact, respectively, with the side chains of Glu-4, Lys-6, and Arg-9 from the peptide to help fix its alignment relative to the active site. In TOP, His-495 and Glu-469 might interact with Glu-4 and Arg-9 from the peptide to help determine the observed cleavage site. The presence of an arginine at position 498 might also play a role in TOP, since with the peptide in the neurolysin registration, that arginine would be positioned opposite Arg-8 and Arg-9 of the peptide, a potentially unfavorable cluster of positive charges. We began attempts to test the role of the four positions in differential specificity by mutating all of the sites in each enzyme to the residues in the corresponding positions of the other enzyme and determining the cleavage site preferences with NT. Hydrolysis of NT by TOP and Neurolysin Mutants—Cleavage site selection by neurolysin and TOP with the NT substrate peptide were determined by separating reaction fragments using reverse phase HPLC (C18 resin) and identifying individual fragments by electrospray ionization mass spectrometry. As expected, wild type TOP hydrolyzed NT between Arg-8 and Arg-9, producing the fragments NT-(1–8) and NT-(9–13), whereas wild type neurolysin cleaved between Pro-10 and Tyr-11, producing fragments NT-(1–10) and NT-(11–13) (Fig. 2). Experimental and expected masses for these fragments are presented in Table 1. (Note that the retention times of the two different C-terminal NT fragments, NT-(9–13) and NT-(11–13), from the C18 reverse phase media are similar, because migration is dominated by the last three hydrophobic/aromatic residues. Mass determination clearly distinguishes between these fragments. The two N-terminal fragments, NT-(1–8) and NT-(1–10), elute at well separated retention times from the C18 media.)TABLE 1Molecular masses of NT fragments produced by wild type TOP, wild type neurolysin, and TOP4EnzymeFragment masses (expected masses)NT1-8NT1-10NT9-13NT11-13NT1-13DaWild type TOP1030.5 (1030.1)661.4 (660.8)1672 (1672.9)Wild type neurolysin1283.6 (1283.4)408.2 (407.5)1672 (1672.9)TOP41283.6 (1283.4)408.2 (407.5) Open table in a new tab Digestion of NT by the TOP four mutant (TOP4) gave products with retention times indistinguishable from those of neurolysin. The masses of the separated fragments were also the same as those produced by neurolysin, demonstrating that, unlike wild type TOP, the mutant enzyme cleaves primarily between Pro-10 and Tyr-11, mimicking neurolysin site recognition. In the reverse case, the neurolysin four mutant (neurolysin 4), produces a hydrolysis pattern indistinguishable from that of wild type TOP, cleaving between Arg-7 and Arg-8. Thus, mutating just the four residues, Glu-469/Arg-470, Met-490/Arg-491, His-495/Asn-496, and Arg-498/Thr-499, is sufficient to completely swap the NT cleavage site recognition of TOP and neurolysin. Interestingly, wild type neurolysin produces a" @default.
• W2080510513 created "2016-06-24" @default.
• W2080510513 creator A5007452748 @default.
• W2080510513 creator A5022211470 @default.
• W2080510513 creator A5035122820 @default.
• W2080510513 creator A5059423838 @default.
• W2080510513 creator A5068888704 @default.
• W2080510513 creator A5090745009 @default.
• W2080510513 date "2007-03-01" @default.
• W2080510513 modified "2023-10-16" @default.
• W2080510513 title "Swapping the Substrate Specificities of the Neuropeptidases Neurolysin and Thimet Oligopeptidase" @default.
• W2080510513 cites W113797329 @default.
• W2080510513 cites W127165557 @default.
• W2080510513 cites W1488354887 @default.
• W2080510513 cites W1533006685 @default.
• W2080510513 cites W1539796472 @default.
• W2080510513 cites W1577408613 @default.
• W2080510513 cites W1595143530 @default.
• W2080510513 cites W1597912030 @default.
• W2080510513 cites W1600835334 @default.
• W2080510513 cites W1607957872 @default.
• W2080510513 cites W1631789991 @default.
• W2080510513 cites W1835492539 @default.
• W2080510513 cites W1854377422 @default.
• W2080510513 cites W1965454305 @default.
• W2080510513 cites W1969234710 @default.
• W2080510513 cites W1969340813 @default.
• W2080510513 cites W1969904017 @default.
• W2080510513 cites W1973432561 @default.
• W2080510513 cites W1976857150 @default.
• W2080510513 cites W1977358971 @default.
• W2080510513 cites W1978901102 @default.
• W2080510513 cites W1984511987 @default.
• W2080510513 cites W1989026158 @default.
• W2080510513 cites W1989806335 @default.
• W2080510513 cites W1989990175 @default.
• W2080510513 cites W1993120181 @default.
• W2080510513 cites W1995017064 @default.
• W2080510513 cites W1996278518 @default.
• W2080510513 cites W1997577244 @default.
• W2080510513 cites W1997852469 @default.
• W2080510513 cites W1997929018 @default.
• W2080510513 cites W2002039326 @default.
• W2080510513 cites W2004091546 @default.
• W2080510513 cites W2004296526 @default.
• W2080510513 cites W2007817980 @default.
• W2080510513 cites W2013083986 @default.
• W2080510513 cites W2014903141 @default.
• W2080510513 cites W2020080086 @default.
• W2080510513 cites W2029768670 @default.
• W2080510513 cites W2031904811 @default.
• W2080510513 cites W2032477994 @default.
• W2080510513 cites W2032870406 @default.
• W2080510513 cites W2033314913 @default.
• W2080510513 cites W2033947177 @default.
• W2080510513 cites W2037650802 @default.
• W2080510513 cites W2037913468 @default.
• W2080510513 cites W2053884159 @default.
• W2080510513 cites W2056681092 @default.
• W2080510513 cites W2062489834 @default.
• W2080510513 cites W2062507404 @default.
• W2080510513 cites W2062819748 @default.
• W2080510513 cites W2071126869 @default.
• W2080510513 cites W2072115646 @default.
• W2080510513 cites W2072686883 @default.
• W2080510513 cites W2072855610 @default.
• W2080510513 cites W2073007718 @default.
• W2080510513 cites W2074752318 @default.
• W2080510513 cites W2076099594 @default.
• W2080510513 cites W2078209952 @default.
• W2080510513 cites W2084254608 @default.
• W2080510513 cites W2091761202 @default.
• W2080510513 cites W2092685120 @default.
• W2080510513 cites W2094102556 @default.
• W2080510513 cites W2107513734 @default.
• W2080510513 cites W2111322358 @default.
• W2080510513 cites W2114474628 @default.
• W2080510513 cites W2137502578 @default.
• W2080510513 cites W2138974986 @default.
• W2080510513 cites W2158707826 @default.
• W2080510513 cites W2175160087 @default.
• W2080510513 cites W2342639517 @default.
• W2080510513 cites W2398525642 @default.
• W2080510513 cites W2064620612 @default.
• W2080510513 doi "https://doi.org/10.1074/jbc.m609897200" @default.
• W2080510513 hasPubMedId "https://pubmed.ncbi.nlm.nih.gov/17251185" @default.
• W2080510513 hasPublicationYear "2007" @default.
• W2080510513 type Work @default.
• W2080510513 sameAs 2080510513 @default.
• W2080510513 citedByCount "25" @default.
• W2080510513 countsByYear W20805105132012 @default.
• W2080510513 countsByYear W20805105132013 @default.
• W2080510513 countsByYear W20805105132015 @default.
• W2080510513 countsByYear W20805105132016 @default.
• W2080510513 countsByYear W20805105132017 @default.
• W2080510513 countsByYear W20805105132018 @default.
• W2080510513 countsByYear W20805105132020 @default.
• W2080510513 countsByYear W20805105132021 @default.

• next