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• W2000483193 abstract "Galactose oxidase is a copper metalloenzyme containing a novel protein-derived redox cofactor in its active site, formed by cross-linking two residues, Cys228 and Tyr272. Previous studies have shown that formation of the tyrosyl-cysteine (Tyr-Cys) cofactor is a self-processing step requiring only copper and dioxygen. We have investigated the biogenesis of cofactor-containing galactose oxidase from pregalactose oxidase lacking the Tyr-Cys cross-link but having a fully processed N-terminal sequence, using both Cu(I) and Cu(II). Mature galactose oxidase forms rapidly following exposure of a pregalactose oxidase-Cu(I) complex to dioxygen (t½ = 3.9satpH7). In contrast, when Cu(II) is used in place of Cu(I) the maturation process requires several hours (t½ = 5.1 h). EDTA prevents reaction of pregalactose oxidase with Cu(II) but does not interfere with the Cu(I)-dependent biogenesis reaction. The yield of cross-link corresponds to the amount of copper added, although a fraction of the pregalactose oxidase protein is unable to undergo this cross-linking reaction. The latter component, which may have an altered conformation, does not interfere with analysis of cofactor biogenesis at low copper loading. The biogenesis product has been quantitatively characterized, and mechanistic studies have been developed for the Cu(I)-dependent reaction, which forms oxidized, mature galactose oxidase and requires two molecules of O2. Transient kinetics studies of the biogenesis reaction have revealed a pH sensitivity that appears to reflect ionization of a protein group (pKa = 7.3) at intermediate pH resulting in a rate acceleration and protonation of an early oxygenated intermediate at lower pH competing with commitment to cofactor formation. These spectroscopic, kinetic, and biochemical results lead to new insights into the biogenesis mechanism. Galactose oxidase is a copper metalloenzyme containing a novel protein-derived redox cofactor in its active site, formed by cross-linking two residues, Cys228 and Tyr272. Previous studies have shown that formation of the tyrosyl-cysteine (Tyr-Cys) cofactor is a self-processing step requiring only copper and dioxygen. We have investigated the biogenesis of cofactor-containing galactose oxidase from pregalactose oxidase lacking the Tyr-Cys cross-link but having a fully processed N-terminal sequence, using both Cu(I) and Cu(II). Mature galactose oxidase forms rapidly following exposure of a pregalactose oxidase-Cu(I) complex to dioxygen (t½ = 3.9satpH7). In contrast, when Cu(II) is used in place of Cu(I) the maturation process requires several hours (t½ = 5.1 h). EDTA prevents reaction of pregalactose oxidase with Cu(II) but does not interfere with the Cu(I)-dependent biogenesis reaction. The yield of cross-link corresponds to the amount of copper added, although a fraction of the pregalactose oxidase protein is unable to undergo this cross-linking reaction. The latter component, which may have an altered conformation, does not interfere with analysis of cofactor biogenesis at low copper loading. The biogenesis product has been quantitatively characterized, and mechanistic studies have been developed for the Cu(I)-dependent reaction, which forms oxidized, mature galactose oxidase and requires two molecules of O2. Transient kinetics studies of the biogenesis reaction have revealed a pH sensitivity that appears to reflect ionization of a protein group (pKa = 7.3) at intermediate pH resulting in a rate acceleration and protonation of an early oxygenated intermediate at lower pH competing with commitment to cofactor formation. These spectroscopic, kinetic, and biochemical results lead to new insights into the biogenesis mechanism. Galactose oxidase (GAOX) 1The abbreviations used are: GAOX, galactose oxidase; pre-GAOX, galactose oxidase pre-enzyme lacking leader peptide and cofactor; AGAOX, oxidized mature cofactor-containing galactose oxidase; αMF, S. cerevisiae α-mating factor leader peptide; MOPS, 3-(N-morpholino)propanesulfonic acid; MES, 2-(N-morpholino)ethanesulfonic acid; CHES, 2-(N-cyclohexylamino)ethanesulfonic acid; DTNB, 5,5′-dithiobis-(2-nitrobenzoic acid); SKIE, solvent kinetic isotope effect; ET, electron transfer; RR, resonance Raman; WT, wild type.1The abbreviations used are: GAOX, galactose oxidase; pre-GAOX, galactose oxidase pre-enzyme lacking leader peptide and cofactor; AGAOX, oxidized mature cofactor-containing galactose oxidase; αMF, S. cerevisiae α-mating factor leader peptide; MOPS, 3-(N-morpholino)propanesulfonic acid; MES, 2-(N-morpholino)ethanesulfonic acid; CHES, 2-(N-cyclohexylamino)ethanesulfonic acid; DTNB, 5,5′-dithiobis-(2-nitrobenzoic acid); SKIE, solvent kinetic isotope effect; ET, electron transfer; RR, resonance Raman; WT, wild type. (EC 1.1.3.9) is a secretory fungal copper metalloenzyme that generates hydrogen peroxide in the extracellular space by oxidizing simple alcohols and subsequently reducing dioxygen to H2O2 (1Avigad G. Amaral D. Asensio C. Horecker B.L. J. Biol. Chem. 1962; 237: 2736-2743Abstract Full Text PDF PubMed Google Scholar, 2Whittaker J.W. Adv. Protein Chem. 2002; 60: 1-50Crossref PubMed Scopus (64) Google Scholar, 3Kosman D.J. Lontie R. Copper Proteins and Copper Enzymes. CRC Press, Inc., Boca Raton, FL1984: 1-26Google Scholar, 4Hamilton G.A. Spiro T.G. Copper Proteins. Wiley Interscience, New York1981: 193-218Google Scholar). Together with a closely related enzyme, glyoxal oxidase (5Whittaker M.M. Kersten P.J. Nakamura N. Sanders-Loehr J. Schweizer E.S. Whittaker J.W. J. Biol. Chem. 1996; 271: 681-687Abstract Full Text Full Text PDF PubMed Scopus (190) Google Scholar, 6Kersten P.J. Proc. Natl. Acad. Sci. U. S. A. 1990; 87: 2936-2940Crossref PubMed Scopus (171) Google Scholar), galactose oxidase represents a family of radical-copper oxidases defined by the presence of an unusual free radical-coupled copper active site (comprising a free radical associated with a redox-active metal ion) that functions as a two-electron redox unit in substrate oxidation and O2 reduction (7Whittaker M.M. Whittaker J.W. J. Biol. Chem. 1988; 263: 6074-6080Abstract Full Text PDF PubMed Google Scholar, 8Whittaker J.W. Metal Ions Biol. Syst. 1994; 30: 315-360Google Scholar). These free radical enzymes (9Stubbe J. van der Donk W.A. Chem. Rev. 1998; 98: 705-762Crossref PubMed Scopus (1349) Google Scholar, 10Frey P.A. Curr. Opin. Chem. Biol. 1997; 1: 347-356Crossref PubMed Scopus (36) Google Scholar) generate catalytic free radicals by reversible oxidation of a tyrosyl side chain in the protein (11Whittaker M.M. Whittaker J.W. J. Biol. Chem. 1990; 265: 9610-9613Abstract Full Text PDF PubMed Google Scholar). X-ray crystallography has revealed that the active site of galactose oxidase (Fig. 1) contains a novel post-translational modification, a covalent bond between Cys228 and Tyr272 creating a new, thioetherbridged cross-linked amino acid, tyrosyl-cysteine (Tyr-Cys), without addition of exogenous atoms (12Ito N. Phillips S.E.V. Stevens C. Ogel Z.B. McPherson M.J. Keen J.N. Yadav K.D.S. Knowles P.F. Nature. 1991; 350: 87-90Crossref PubMed Scopus (685) Google Scholar, 13Ito N. Phillips S.E.V. Yadav K.D.S. Knowles P.F. J. Mol. Biol. 1994; 238: 794-814Crossref PubMed Scopus (286) Google Scholar) (Scheme 1). Spectroscopic and chemical modeling studies have demonstrated that this Tyr-Cys side chain is, in fact, the free radical site in the protein (15Babcock G.T. El-Deeb M.K. Sandusky P.O. Whittaker M.M. Whittaker J.W. J. Am. Chem. Soc. 1992; 114: 3727-3734Crossref Scopus (141) Google Scholar, 16McGlashin M.L. Eads D.D. Spiro T.G. Whittaker J.W. J. Phys. Chem. 1995; 99: 4918-4922Crossref Scopus (85) Google Scholar, 17Gerfen G.A. Bellew B. Griffin R. Singel D. Ekberg C.A. Whittaker J.W. J. Phys. Chem. 1996; 100: 16739-16748Crossref Scopus (64) Google Scholar, 18Whittaker M.M. Chuang Y.Y. Whittaker J.W. J. Am. Chem. Soc. 1993; 115: 10029-10035Crossref Scopus (106) Google Scholar, 19Itoh S. Takayama S. Arakawa R. Furuta A. Komatsu M. Ishida A. Takamuku S. Fukuzumi S. Inorg. Chem. 1997; 36: 1407-1416Crossref PubMed Scopus (132) Google Scholar). Characterization of the radical copper catalytic motif in galactose oxidase is complicated by the presence of multiple species in the as-isolated enzyme, including a significant fraction of apoenzyme, distinct forms of the metal- and cofactor-containing holoenzyme differing essentially in the number of electrons in the active site, as well as a portion of holoenzyme that is unable to generate a free radical complex and therefore does not contribute to catalytic activity (2Whittaker J.W. Adv. Protein Chem. 2002; 60: 1-50Crossref PubMed Scopus (64) Google Scholar, 7Whittaker M.M. Whittaker J.W. J. Biol. Chem. 1988; 263: 6074-6080Abstract Full Text PDF PubMed Google Scholar, 8Whittaker J.W. Metal Ions Biol. Syst. 1994; 30: 315-360Google Scholar).Scheme 1View Large Image Figure ViewerDownload Hi-res image Download (PPT)X-ray structural studies are revealing cross-linked amino acid side chains in the active sites of a number of other redox metalloenzymes, including cytochrome c oxidase (20Ostermeier C. Harrenga A. Ermler U. Michel H. Proc. Natl. Acad. Sci. U. S. A. 1997; 94: 10547-10553Crossref PubMed Scopus (711) Google Scholar, 21Buse G. Soulimane T. Dewor M. Meyer H.E. Bluggel M. Protein Sci. 1999; 8: 985-990Crossref PubMed Scopus (107) Google Scholar, 22Tsukihara T. Aoyama H. Yamashita E. Tomizaki T. Yamaguchi H. Shinazawa-Itoh K. Nakashima R. Yaono R. Yoshikawa S. Science. 1996; 272: 1136-1144Crossref PubMed Scopus (1900) Google Scholar) (tyrosyl-histidine), catechol oxidase, hemocyanin (23Cuff M.E. Miller K.I. van Holde K.E. Hendrickson W.A. J. Mol. Biol. 1998; 278: 855-870Crossref PubMed Scopus (340) Google Scholar, 24Gielens C. De Geest N. Xin X.Q. Devreese B. Van Beeumen J. Preaux G. Eur. J. Biochem. 1997; 248: 879-888Crossref PubMed Scopus (51) Google Scholar, 25Klabunde T. Eicken C. Sacchettini J.C. Krebs B. Nat. Struct. Biol. 1998; 5: 1084-1090Crossref PubMed Scopus (743) Google Scholar) (histidyl-cysteine), and methylamine dehydrogenase (26Chen L. Doi M. Durley R.C. Chistoserdov A.Y. Lidstrom M.E. Davidson V.L. Mathews F.S. J. Mol. Biol. 1998; 276: 131-149Crossref PubMed Scopus (99) Google Scholar, 27Davidson V.L. Adv. Protein Chem. 2001; 58: 95-140Crossref PubMed Scopus (93) Google Scholar) (tryptophanyl-tryptophan). The origins and functions of these specialized elements of protein structure are just beginning to be investigated in detail. Formation of the tryptophanyl-tryptoquinone catalytic cofactor in methylamine dehydrogenase appears to involve processing by ancillary enzymes (28Graichen M.E. Jones L.H. Sharma B.V. van Spanning R.J. Hosler J.P. Davidson V.L. J. Bacteriol. 1999; 181: 4216-4222Crossref PubMed Google Scholar). In contrast, galactose oxidase cofactor biogenesis appears to be a self-processing event requiring only copper and O2 (29Rogers M.S. Baron A.J. McPherson M.J. Knowles P.F. Dooley D.M. J. Am. Chem. Soc. 2000; 122: 990-991Crossref Scopus (68) Google Scholar). Galactose oxidase is distinct from the other examples in being a secretory protein, which means that its release in functional form involves at least three processing steps as follows: cleavage of the prepro leader sequence that directs translocation of the nascent polypeptide chain into the secretory pathway, metal binding, and cofactor biogenesis. The structure of a partially processed precursor form of the protein, containing the 17-amino acid N-terminal prosequence leader peptide but lacking both copper and cofactor, has been reported recently (30Firbank S.J. Rogers M.S. Wilmot C.M. Dooley D.M. Halcrow M.A. Knowles P.F. McPherson M.J. Phillips S.E.V. Proc. Natl. Acad. Sci. U. S. A. 2001; 98: 12932-12937Crossref PubMed Scopus (100) Google Scholar). The active site region of the unprocessed protein in the crystal exhibits large amplitude displacements of critical active site residues, reflecting a significant degree of conformational flexibility in the incompletely processed protein. Covalent modification of the active site cysteine to a sulfenic acid (Cys228-SOH) observed in the crystal may reflect oxidative damage to the protein. The precursor form of galactose oxidase has been shown to form spontaneously the cofactor in the presence of Cu(II) and dioxygen (29Rogers M.S. Baron A.J. McPherson M.J. Knowles P.F. Dooley D.M. J. Am. Chem. Soc. 2000; 122: 990-991Crossref Scopus (68) Google Scholar). The successful heterologous expression of galactose oxidase in Pichia pastoris using non-native leader peptide to direct secretion indicates that the native prepro signal is not strictly required for either secretion of the protein or biogenesis of functional cofactor (31Whittaker M.M. Whittaker J.W. Protein Expression Purif. 2000; 20: 105-111Crossref PubMed Scopus (59) Google Scholar). Therefore, in this work we have investigated in vitro biogenesis of the galactose oxidase redox cofactor, making use of recombinant protein containing the fully processed N terminus (no signal sequence) and lacking the thioether bond between Cys228 and Tyr272. Extensive and quantitative analysis of the cross-linking reaction and thorough characterization of the biogenesis product (by SDS-PAGE, activity measurements, optical absorption spectroscopy, and metal quantitation) is now leading to new insight into the mechanism of cofactor formation in galactose oxidase.EXPERIMENTAL PROCEDURESBiological Materials—P. pastoris X33 (32Higgins D.R. Cregg J.M. Methods Mol. Biol. 1998; 103: 1-15PubMed Google Scholar) was obtained from Invitrogen. Recombinant galactose oxidase was purified from high density methanol fermentation medium (33Stratton J. Chiruvolu V. Meagher M. Methods Mol. Biol. 1998; 103: 107-120PubMed Google Scholar) of a P. pastoris transformant prepared by multicopy chromosomal integration of an expression cassette comprising the pPICZ Zeocin-selection plasmid (Invitrogen) linearized within the AOX1 promoter by digestion with PmeI. The coding region of the expression cassette contains a 5′-nucleotide sequence coding for either the Aspergillus niger glucoamylase leader peptide (Gla) or the Saccharomyces cerevisiae α-mating factor leader peptide (αMF) spliced with the cDNA sequence corresponding to the secreted galactose oxidase protein. The galactose oxidase coding sequence was modified for expression of GAOX mutational variants (C228G, Y272G) using Stratagene (La Jolla, CA) QuikChange™ site-directed mutagenesis kit, and the protein was expressed and purified as described previously. For secretion of the unprocessed, cofactor-free pregalactose oxidase protein, the transformant was grown as described previously (31Whittaker M.M. Whittaker J.W. Protein Expression Purif. 2000; 20: 105-111Crossref PubMed Scopus (59) Google Scholar) except that the copper content of the medium was reduced to 15% of the amount described for the PTM4 trace metals supplement in the earlier work (33Stratton J. Chiruvolu V. Meagher M. Methods Mol. Biol. 1998; 103: 107-120PubMed Google Scholar). The other modifications to fermentation conditions reported previously (31Whittaker M.M. Whittaker J.W. Protein Expression Purif. 2000; 20: 105-111Crossref PubMed Scopus (59) Google Scholar) were the elimination of casamino acids from the methanol induction phase and the exclusion of copper from the methanol feed-stock during expression. Pregalactose oxidase was purified as described previously (31Whittaker M.M. Whittaker J.W. Protein Expression Purif. 2000; 20: 105-111Crossref PubMed Scopus (59) Google Scholar) except that 2 mm EDTA was present in all buffer solutions. For biogenesis studies, the protein was passed through a gel filtration column to remove EDTA. Protocatechuate 3,4-dioxygenase was isolated from Brevibacterium fuscum as described previously (34Whittaker J.W. Lipscomb J.D. Kent T.A. Munck E. J. Biol. Chem. 1984; 259: 4466-4475Abstract Full Text PDF PubMed Google Scholar).Reagents—MOPS, MES, CHES, 5,5′-dithiobis-(2-nitrobenzoic acid) (DTNB), l-cysteine, 1-O-methyl-α-d-galactopyranoside, and EDTA were obtained from Sigma. Acetonitrile, deuterium oxide (99.9 atom % 2H), and tetrakis(acetonitrile) copper(I) hexafluorophosphate [Cu(I) (CH3CN)4·PF6] were purchased from Aldrich. Guanidinium hydrochloride was from Pierce, and potassium ferricyanide was from Fluka (White Plains, NY).Biochemical Methods—Protein concentrations of purified galactose oxidase and pregalactose oxidase were determined by optical absorption measurements, using the molar extinction coefficient at 280 nm (ϵ280 = 1.05 × 105m–1 cm–1) as reported previously (35Tressel P.S. Kosman D.J. Methods Enzymol. 1982; 89: 163-171Crossref PubMed Scopus (41) Google Scholar). Proteins resolved by SDS-PAGE (Bio-Rad ready-gels) were stained with GelCode Blue™ staining solution (Pierce). Gels were digitized using a scanner and analyzed with tnimage Measurement and Analysis program (36Nelson T.J. Tnimage Scientific Image Measurement and Analysis Lab Manual. The Johns Hopkins University, Baltimore, MD2000Google Scholar). Strip densitometric scan data were further analyzed using the line shape deconvolution routines of the Grams spectral analysis program (Galactic Industries Corp., Salem, NH). N-terminal sequence analysis of purified pregalactose oxidase was performed by Debra A. McMillen at the Biotechnology Laboratory, Institute of Molecular Biology, University of Oregon.Quantitation of free sulfhydryl groups in pregalactose oxidase, galactose oxidase, and variants was done using the DTNB test on protein in 4 m guanidinium hydrochloride using cysteine as standard (37Riddles P.W. Blakely R.L. Zerner B. Anal. Biochem. 1979; 94: 75-81Crossref PubMed Scopus (918) Google Scholar). Samples were heated in a water bath at 100 °C for 1 min and cooled on ice before addition of DTNB. Deblocking of cysteine sulfenic acid groups was performed as described (38You K.-S. Benitz L.V. McConachie W.A. Allison W.S. Biochim. Biophys. Acta. 1975; 384: 317-330Crossref PubMed Scopus (37) Google Scholar, 39Claiborne A. Yeh J.I. Mallett T.C. Luba J. Crane III, E.J. Charrier V. Pasonage D. Biochemistry. 1999; 38: 15047-15416Crossref Scopus (457) Google Scholar, 40Allison W.S. Acc. Chem. Res. 1976; 9: 293-299Crossref Scopus (250) Google Scholar). Briefly, protein was incubated with 20 mml-ascorbate in 50 mm MES buffer, pH 5.3, for 0.5 h, and the reductant was removed by gel filtration.Biogenesis of the galactose oxidase cofactor from pregalactose oxidase and Cu(I) was conducted under argon purge in 20 mm MOPS, pH 7. Tetrakis(acetonitrile) Cu(I) hexafluorophosphate [Cu(I)(CH3CN)4·PF6] was dissolved in anaerobic acetonitrile immediately before addition of an aliquot of this solution to argon-purged pregalactose oxidase. After mixing, the pregalactose oxidase-Cu(I) solution was rapidly purged with pure oxygen. Quantitation of the oxygen stoichiometry for cofactor biogenesis was performed by adding aliquots of air-saturated buffer to a mixture containing pregalactose oxidase and a substoichiometric amount of Cu(I). The pregalactose oxidase-Cu(I) complex was prepared by combining 150 nmol of Cu(I)(CH3CN)4·PF6 with 600 nmol of pregalactose oxidase in a 3-ml anaerobic cuvette under argon. The optical absorption of the stirred solution was monitored at 445 nm.The mutual stability of pre-GAOXCu(I) and fully oxidized mature GAOX in a mixture containing both was determined by sequential preparation of a pre-GAOXCu(I) complex, addition of oxidized GAOX, and O2 in that order. Copper(I) acetonitrile (60 nmol, 0.6 eq) was added to an anaerobic solution of pre-GAOX (100 μm, 1 ml) under argon. An anaerobic solution of fully oxidized mature GAOX (AGAOX) (60 nmol in 50 μl) was added, and the optical absorption spectrum was recorded and monitored at 445 nm. The cuvette was then purged with O2 gas and the optical spectrum recorded.Copper analyses were performed using a Varian Instruments SpectrAA atomic absorption spectrometer equipped with a GTA 96 graphite furnace. Galactose oxidase activity was measured by oxygen uptake in a Clark-type oxygen electrode. The assay mixture contained 50 mmO-methyl-α-d-galactopyranoside, 2 mm K3Fe(CN)6, and 50 mm KH2PO4, pH 7. The reaction was monitored at 25 °C, and the electrode was calibrated using the protocatechuic acid/protocatechuate dioxygenase reaction (41Whittaker M.M. Ballou D.P. Whittaker J.W. Biochemistry. 1998; 37: 8426-8436Crossref PubMed Scopus (133) Google Scholar).Spectroscopic Methods—Optical absorption spectra were measured using a Varian Instruments Cary 5E UV-visible near infrared absorption spectrophotometer. Electron paramagnetic resonance spectra were recorded on a Bruker E500 X-Band EPR spectrometer with a Super X microwave bridge and SHQ resonator equipped with a nitrogen flow cryostat. EPR signal quantitation was performed using Cu(II) perchlorate spin standard. First derivative solution EPR spectra were simulated using the program sim15 (Quantum Chemistry Program Exchange QCPE265). Solution EPR spectra were recorded for samples in a quartz flat cell (Wilmad Glass, Buena, NJ) at 305 K. Resonance Raman spectra were collected with a custom McPherson 2061/207 spectrograph (0.67-m focal length, 600 groove grating, 7 cm–1 spectral resolution) using a Coherent Inova 302 krypton laser (413 nm), a Kaiser optical super-notch filter, and a Princeton Instruments (LN-1100PB) liquid N2-cooled CCD detector. Spectra were obtained for samples in glass capillaries at 300 K using a 90 ° scattering geometry and a 20-min accumulation time. Sample integrity was verified by the observation of the same absorption spectrum before and after laser irradiation.Transient Kinetics—The rate of formation of mature galactose oxidase from pregalactose oxidase and Cu(I) was measured using a Biologic SFM-300 rapid mixing stopped-flow module connected to an OLIS RSM-1000 rapid scanning monochromator via fiber optic light pipes. Prior to the rapid mixing experiment, the stopped-flow system was scrubbed free of oxygen using 50 ml of protocatechuic acid/protocatechuate dioxygenase mixture in 50 mm Tris, pH 8, for at least 24 h (42Patil P.V. Ballou D.P. Anal. Biochem. 2000; 286: 187-192Crossref PubMed Scopus (91) Google Scholar). All syringes were periodically emptied and refilled with the scrubbing reagents. During this period the syringe chamber was purged with nitrogen gas, and the circulating water bath reservoir was sparged with nitrogen. Collars fitted around the syringe ports on the Biologic SFM-300 head permitted a continuous argon purge of these points. Anaerobic solutions delivered to the syringe block were prepared in gas-tight tonometers. Cu(I)(CH3CN)4·PF6 in acetonitrile was added to an anaerobic solution of pregalactose oxidase in 20 mm MOPS, pH 7, that was subsequently transferred anaerobically to an argon-purged tonometer using a double-ended catheter needle. The cofactor biogenesis reaction was initiated by mixing this solution with oxygenated buffer in the stopped-flow system, monitoring the formation of fully oxidized cofactor-containing mature galactose oxidase. The pH sensitivity of the biogenesis reaction was investigated by shooting pregalactose oxidase/Cu(I) (prepared anaerobically in buffer-free water) against airsaturated 40 mm buffer solutions in the SFM-300 syringe driver. The buffer solutions used are as follows: pH 5.0–5.5, MES; pH 6.0–6.5, MES/MOPS mixture; pH 7.0, MOPS; pH 7.5–8.0, MOPS/CHES mixture; pH 8.5, CHES. Solvent kinetic isotope (SKIE) measurements were performed by shooting pregalactose oxidase/Cu(I) prepared as above in either H2O or D2O (containing 0.5 mm MOPS, pL = 7) against airsaturated buffers (50 mm before mixing) in H2O or D2O, adjusted to the same pL value by volumetric mixing of components. Global data analysis and kinetic model evaluation was performed using the program Specfit/32 (Spectrum Software Associates, Marlborough, MA).RESULTSProduction of Pregalactose Oxidase—P. pastoris transformants containing galactose oxidase cDNA linked to either A. niger glucoamylase leader peptide or S. cerevisiae αMF leader peptide coding sequences efficiently secrete mature galactose oxidase under methanol-induced expression in complete medium (31Whittaker M.M. Whittaker J.W. Protein Expression Purif. 2000; 20: 105-111Crossref PubMed Scopus (59) Google Scholar). When copper supplementation was reduced in the glycerol batch growth phase and eliminated completely in the induction phase of high density methanol fermentation, the transformants produced pregalactose oxidase lacking the thioether cross-link between Cys228 and Tyr272 (Fig. 1) but exhibiting the N-terminal sequence (ASAPI) of the authentic mature protein. The yield of purified pregalactose oxidase was ∼200 mg/liter for a 5-liter fermentation culture harvested 1 day after induction. The background galactose oxidase activity of the purified pregalactose oxidase was about 0.1% (∼ 0.7 μmol of O2/mg of protein at 25 °C) of the recombinant WT galactose oxidase. Analysis of the sulfhydryl content of pregalactose oxidase using the DTNB assay (Table I) shows that two representative preparations of pre-enzyme contain approximately twice as many free SH groups as mature WT galactose oxidase, confirming the presence of an additional, predominantly unblocked cysteine residue (Cys228) in these preparations. The amount of free sulfhydryl varies somewhat between preparations.Table IAnalysis of free sulfhydryl content of galactose oxidase variantsVariantmol SH/mol proteinaSulfhydryl content determined by DTNB assay as described under “Experimental Procedures.”WT apoGAOX0.76C228G GAOX0.89Y272G GAOX1.49Pre-GAOX (A)bDetermination of free sulfhydryl content for two independent pre-GAOX preparations.1.68Pre-GAOX (B)bDetermination of free sulfhydryl content for two independent pre-GAOX preparations.1.52a Sulfhydryl content determined by DTNB assay as described under “Experimental Procedures.”b Determination of free sulfhydryl content for two independent pre-GAOX preparations. Open table in a new tab Detection of Cross-linked Product by SDS-PAGE—Mature, cross-linked cofactor-containing galactose oxidase shows an altered mobility on SDS-PAGE gels (Fig. 2, lane 6) and is resolved from unmodified pregalactose oxidase (Fig. 2, lane 1), as reported previously (29Rogers M.S. Baron A.J. McPherson M.J. Knowles P.F. Dooley D.M. J. Am. Chem. Soc. 2000; 122: 990-991Crossref Scopus (68) Google Scholar). Mature galactose oxidase appeared following addition of Cu(I)(CH3CN)4·PF6 to pregalactose oxidase under anaerobic conditions followed by oxygen gas purging (Fig. 2, lanes 2–5). The amount of mature product formed was roughly proportional to Cu(I) added up to about 0.8 eq (Table I, pre-GAOX(A)). Lower conversion was observed in some preparations (Table I, pre-GAOX(B)), in which the amount of cross-linked product was proportional to Cu(I) added only up to about 0.5 eq. Thus, the upper limit to conversion appears to vary somewhat between pre-enzyme preparations. Similar results were obtained by addition of Cu(II)SO4 to pre-galactose oxidase in the presence of oxygen (Fig. 2, lane 7). Varying the sample concentration (from micromolar to millimolar) did not change the yield, and the protein began to precipitate when more than 1 eq of copper was added. Unmodified pre-GAOX appears to be more sensitive to precipitation in excess copper than the mature GAOX protein. Treating pre-GAOX with ascorbic acid at low pH (a method for deblocking cysteine sulfenic acid groups, Cys-SOH) did not increase the yield of mature GAOX. Other attempts (varying salt concentration, pH, buffer, addition of superoxide and hydrogen peroxide scavengers, and repeating the Cu(I) treatment on the initially formed biogenesis product) also failed to increase the upper limit of conversion to the mature GAOX product.Fig. 2SDS-PAGE analysis of cross-link formation in the cofactor biogenesis reaction.Top, GelCode™-stained 12% SDS-PAGE (loaded with 0.5 μg of protein in each lane) resolving uncross-linked pre-GAOX and cross-linked mature GAOX product. Reaction conditions are as described under “Experimental Procedures” and in Table II. Lane 1, pre-GAOX; lane 2, reaction 1; lane 3, reaction 2; lane 4, reaction 3; lane 5, reaction 4; lane 6, mature GAOX; lane 7, reaction 5. The distinct electrophoretic mobility of pre-GAOX (a) and mature GAOX (b) is indicated at the left. Bottom, strip scan densitometric traces for SDS-PAGE data. Individual lanes 1–7 were analyzed using the strip densitometry routine of tnimage analysis software as described under “Experimental Procedures.” The positions of the pre-GAOX (a) and mature GAOX (b) protein standards are indicated.View Large Image Figure ViewerDownload Hi-res image Download (PPT)Densitometric analysis of the scanned gel (Fig. 2, bottom) provides quantitative information on the yield of mature, crosslinked GAOX in the biogenesis reaction (Table II). Gaussian resolution of the individual protein bands in the stained SDS-PAGE is reported in Table II. The yield of cross-link correlates with the amount of copper added up to about 0.8 eq. The upper band on the gel (a), corresponding to pre-GAOX, is broader than that associated with the mature protein (b), and the broadening increases with the age of the sample.Table IIDensitometric gel scan analysis of cross-link formation in the in vitro biogenesis reactionLaneReaction/sampleaReaction conditions are as described under “Experimental Procedures.”Cu mol/mol proteinbReactions 1-4" @default.
• W2000483193 created "2016-06-24" @default.
• W2000483193 creator A5017786515 @default.
• W2000483193 creator A5044320588 @default.
• W2000483193 date "2003-06-01" @default.
• W2000483193 modified "2023-09-26" @default.
• W2000483193 title "Cu(I)-dependent Biogenesis of the Galactose Oxidase Redox Cofactor" @default.
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