Cysteine 707 Is Involved in the Dehydrogenase Active Site of Rat 10-Formyltetrahydrofolate Dehydrogenase
1995; Elsevier BV; Volume: 270; Issue: 2 Linguagem: Inglês
10.1074/jbc.270.2.519
ISSN1083-351X
AutoresSergey A. Krupenko, Conrad Wagner, Robert Cook,
Tópico(s)Cancer, Hypoxia, and Metabolism
ResumoThe enzyme, 10-formyltetrahydrofolate dehydrogenase (10-FTHFDH) (EC 1.5.1.6) catalyzes both the NADP + -dependent oxidation of 10-formyltetrahydrofolate to tetrahydrofolate and CO2 and the NADP + -independent hydrolysis of 10-formyltetrahydrofolate to tetrahydrofolate and formate. The COOH-terminal domain of the 10-FTHFDH (residues 417-902) shows a 46% identity with a series of NAD + -dependent aldehyde dehydrogenases (EC 1.2.1.3). All known members of the aldehyde dehydrogenase family and 10-FTHFDH have a strictly conserved cysteine (Cys-707 for 10-FTHFDH), which has been predicted to be at the active site of these enzymes. Rat liver 10-FTHFDH was expressed in a baculovirus system, and site-directed mutagenesis has been used to study the role of cysteine 707 in the activity of 10-FTHFDH. 10-FTHFDH with alanine substituted for cysteine at position 707 had no dehydrogenase activity, while hydrolase activity and binding of NADP + were unchanged. Light scattering analysis revealed that wild type and mutant 10-FTHFDH exist as tetramers. We conclude that cysteine 707 is directly involved in the active site of 10-FTHFDH responsible for dehydrogenase activity, and there is a separate site for the hydrolase activity. The enzyme, 10-formyltetrahydrofolate dehydrogenase (10-FTHFDH) (EC 1.5.1.6) catalyzes both the NADP + -dependent oxidation of 10-formyltetrahydrofolate to tetrahydrofolate and CO2 and the NADP + -independent hydrolysis of 10-formyltetrahydrofolate to tetrahydrofolate and formate. The COOH-terminal domain of the 10-FTHFDH (residues 417-902) shows a 46% identity with a series of NAD + -dependent aldehyde dehydrogenases (EC 1.2.1.3). All known members of the aldehyde dehydrogenase family and 10-FTHFDH have a strictly conserved cysteine (Cys-707 for 10-FTHFDH), which has been predicted to be at the active site of these enzymes. Rat liver 10-FTHFDH was expressed in a baculovirus system, and site-directed mutagenesis has been used to study the role of cysteine 707 in the activity of 10-FTHFDH. 10-FTHFDH with alanine substituted for cysteine at position 707 had no dehydrogenase activity, while hydrolase activity and binding of NADP + were unchanged. Light scattering analysis revealed that wild type and mutant 10-FTHFDH exist as tetramers. We conclude that cysteine 707 is directly involved in the active site of 10-FTHFDH responsible for dehydrogenase activity, and there is a separate site for the hydrolase activity. INTRODUCTION10-Formyltetrahydrofolate dehydrogenase (10-FTHFDH) ( 1The abbreviations used are: 10-FTHFDH10-formyltetrahydrofolate dehydrogenase10-HCO-H4PteGlu10-formyltetrahydrofolateH4PteGlutetrahydrofolate10-FDDF10-formyl-5,8-dideazafolate2-ME2-mercaptoethanolPCRpolymerase chain reaction.) (EC 1.5.1.6) catalyzes both the NADP + -dependent oxidation of 10-formyltetrahydrofolate (10-HCO-H4PteGlu) to tetrahydrofolate and CO2 and the NADP + -independent hydrolysis of 10-HCO-H4PteGlu to tetrahydrofolate and formate(1Kutzbach C. Stokstad E.L.R. Methods Enzymol. 1971; 18B: 793-798Crossref Scopus (29) Google Scholar, 2Rios-Orlandi E.M. Zarkadas C.G. MacKenzie R.E. Biochim. Biophys. Acta. 1986; 871: 24-35Crossref PubMed Scopus (36) Google Scholar, 3Scrutton M.C. Beis I. Biochem. J. 1979; 177: 833-846Crossref PubMed Scopus (28) Google Scholar). The amino-terminal domain of rat liver 10-FTHFDH (residues 1-203) is 24-30% identical to a group of glycinamide ribonucleotide transformylases (EC 2.1.2.1) from different species(4Cook R.J. Lloyd R.S. Wagner C. J. Biol. Chem. 1991; 266: 4965-4973Abstract Full Text PDF PubMed Google Scholar). The carboxyl-terminal domain (residues 417-902) of 10-FTHFDH has 46% identity with a series of NAD + -dependent aldehyde dehydrogenases (EC 1.2.1.3), and the enzyme is able to oxidize aldehydes(4Cook R.J. Lloyd R.S. Wagner C. J. Biol. Chem. 1991; 266: 4965-4973Abstract Full Text PDF PubMed Google Scholar).All known aldehyde dehydrogenases contain a conserved cysteine, which, in the case of 10-FTHFDH, is Cys-707(4Cook R.J. Lloyd R.S. Wagner C. J. Biol. Chem. 1991; 266: 4965-4973Abstract Full Text PDF PubMed Google Scholar, 5Hempel J. Nicholas H. Lindahl R. Protein Sci. 1993; 2: 1890-1900Crossref PubMed Scopus (166) Google Scholar). The conserved cysteine is presumed to act as a nucleophile in the formation of an enzyme-linked thiohemiacetal intermediate(6Weiner H. Farres J. Wang T.T.Y. Cunningham S.J. Zheng C.-F. Ghenbot G. Weiner H. Wermuth B. Crabb D.W. Enzymology and Molecular Biology of Carbonyl Metabolism. Vol. 3. Plenum Press, New York1991: 13-17Google Scholar, 7Blatter E.E. Abriola D.P. Pietruszko R. Biochem. J. 1992; 282: 353-360Crossref PubMed Scopus (39) Google Scholar). A number of different approaches may be used to identify the amino acid at the active site of an enzyme. Using an affinity label, Blatter et al.(7Blatter E.E. Abriola D.P. Pietruszko R. Biochem. J. 1992; 282: 353-360Crossref PubMed Scopus (39) Google Scholar) found that Cys-302 formed a covalent intermediate with the substrate in human aldehyde dehydrogenase(7Blatter E.E. Abriola D.P. Pietruszko R. Biochem. J. 1992; 282: 353-360Crossref PubMed Scopus (39) Google Scholar). Site-directed mutagenesis confirmed that Cys-302 is essential for enzyme activity of rat liver mitochondrial aldehyde dehydrogenase, whereas Cys-49 and Cys-162 can be changed to alanine without altering enzyme activity(6Weiner H. Farres J. Wang T.T.Y. Cunningham S.J. Zheng C.-F. Ghenbot G. Weiner H. Wermuth B. Crabb D.W. Enzymology and Molecular Biology of Carbonyl Metabolism. Vol. 3. Plenum Press, New York1991: 13-17Google Scholar). Additionally, site-directed mutagenesis experiments have shown that serine 74 is also important for enzyme activity(6Weiner H. Farres J. Wang T.T.Y. Cunningham S.J. Zheng C.-F. Ghenbot G. Weiner H. Wermuth B. Crabb D.W. Enzymology and Molecular Biology of Carbonyl Metabolism. Vol. 3. Plenum Press, New York1991: 13-17Google Scholar). This observation was unexpected because this serine is conserved only in mammalian aldehyde dehydrogenases(5Hempel J. Nicholas H. Lindahl R. Protein Sci. 1993; 2: 1890-1900Crossref PubMed Scopus (166) Google Scholar). It has been shown recently that serine 74 is not involved in aldehyde oxidation but may be involved in NAD + binding(8Rout U.K. Weiner H. Biochemistry. 1994; 33: 8955-8961Crossref PubMed Scopus (17) Google Scholar). In the present study we expressed rat 10-FTHFDH in a baculovirus system and used site-directed mutagenesis to elucidate the role of the highly conserved Cys-707 in the activity of the enzyme.EXPERIMENTAL PROCEDURESMaterials10-Formyl-5,8-dideazafolate (10-FDDF) was obtained from Dr. John B. Hynes, Dept. of Pharmaceutical Chemistry, Medical University of South Carolina. (6R,S)-10-HCO-H4PteGlu was prepared from (6R,S)-5-HCO-H4PteGlu (Sigma) by the method of Rabinowitz(9Rabinowitz J.C. Methods Enzymol. 1963; 6: 814-816Crossref Scopus (101) Google Scholar). Oligonucleotides were synthesized by Research Genetics (Huntsville, AL). Restriction enzymes were purchased from New England BioLabs, Inc. (Beverly, MA) or from Stratagene (La Jolla, CA). Grace's insect cell medium and baculovirus agarose were obtained from Invitrogen (San Diego, CA). Fetal bovine serum was purchased from Intergen (Purchase, NY). Other chemicals were obtained from Sigma.Vector Construction and Enzyme ExpressionA PCR fragment including 277 base pairs of the 5′-coding sequence of 10-FTHFDH from the start codon to a unique NcoI restriction site and having an XbaI restriction site inserted at the 5′-end was synthesized using two oligonucleotides 5′-CCGGCCCATGGGTATGAACTGGCTGCAGAAGG-3′ and 5′-CCGGCTCTAGAATGAAGATTGCAGTAATCGGA-3′ by PCR using a Gene Amp kit (Perkin Elmer, Norwalk, CT). The cDNA of rat liver 10-FTHFDH (4Cook R.J. Lloyd R.S. Wagner C. J. Biol. Chem. 1991; 266: 4965-4973Abstract Full Text PDF PubMed Google Scholar) was subcloned from pBS KS + II into the pVL 1393 baculovirus vector (Invitrogen) through the unique EcoRI restriction site. A fragment between the unique XbaI restriction site of pVL 1393 and the unique NcoI restriction site in the 10-FTHFDH cDNA sequence, including the whole 5′-noncoding sequence of cDNA was removed and replaced with the XbaI-NcoI PCR-generated fragment, containing the coding sequence (Fig. 1). The sequence of the construct was confirmed by the dideoxynucleotide chain termination method (10Sanger F. Nicklen S. Coulson A.R. Proc. Natl. Acad. U. S. A. 1977; 74: 5463-5467Crossref PubMed Scopus (52344) Google Scholar) using a Sequenase DNA sequencing kit (U. S. Biochemical Corp.). This construct was expressed in Sf9 cells, which were grown in monolayer using the MaxBac expression system (Invitrogen) according to the manufacturer's directions.Mutant ConstructionMutation of the 10-FTHFDH was achieved by oligonucleotide-directed mutagenesis. The codon for cysteine, TGC, at position 707 was altered to GCC to produce an alanine-containing mutant. The Transformer site-directed mutagenesis kit (Clontech, Palo Alto, CA), based on the method of Deng and Nickoloff(11Deng W.P. Nickoloff J.A. Anal. Biochem. 1992; 200: 81-88Crossref PubMed Scopus (1079) Google Scholar), was used to introduce the mutation into the pVL 1393-10-FTHFDH construct. Two oligonucleotides were used, the mutagenic oligonucleotide 5′-CAAAGGGGAGAACGCCATTGCGGCAG-3′ for introduction of the mutation in 10-FTHFDH sequence and a selection oligonucleotide 5′-GAATTCCGGAGAGGCCTCTGCAGATC-3′ for conversion of one unique restriction site, XmaIII to StuI, which was also unique, in pVL 1393. The mutagenic experiments were carried out according to the Clontech protocol. Introduction of the mutation was confirmed by sequencing(10Sanger F. Nicklen S. Coulson A.R. Proc. Natl. Acad. U. S. A. 1977; 74: 5463-5467Crossref PubMed Scopus (52344) Google Scholar). The mutant construct was expressed in Sf9 cells similar to that for wild type protein.Analysis of Recombinant 10-FTHFDHSDS-electrophoresis of 10-FTHFDH was done according to the method of Laemmli(12Laemmli U.K. Nature. 1970; 227: 680-685Crossref PubMed Scopus (205953) Google Scholar). Western immunoblot analysis was performed as described by Burnette (13Burnette W.N. Anal. Biochem. 1981; 112: 195-203Crossref PubMed Scopus (5863) Google Scholar) with rabbit polyclonal antiserum against pure rat liver 10-FTHFDH and goat anti-rabbit IgG conjugated to alkaline phosphatase (Bio-Rad)(14Leary J.J. Brigati D.J. Ward D.C. Proc. Natl. Acad. Sci. U. S. A. 1983; 80: 4045-4049Crossref PubMed Scopus (893) Google Scholar). The mutant protein showed the same level of expression as wild type recombinant 10-FTHFDH. Analysis of culture medium and cell extract revealed that most of the mutant enzyme was released from cells into the culture medium, similar to the wild type recombinant enzyme, possibly due to lysis of the cells, which occurs in the later post-infection period(15Luckow V.A. Summers M.D. Bio/Technology. 1988; 6: 47-55Crossref Scopus (865) Google Scholar). More than 80% of the total enzyme was found in the culture medium. Immunoblotting with specific anti-10-FTHFDH antiserum showed only one major band corresponding to the enzyme itself, indicating no proteolytic degradation of the enzyme during production.Purification of Recombinant 10-FTHFDHThe enzyme was purified from the culture medium by affinity chromatography on a column of Sepharose-5-formyltetrahydrofolate(16Cook R.J. Wagner C. Biochemistry. 1982; 21: 4427-4434Crossref PubMed Scopus (31) Google Scholar, 17Cook R.J. Wagner C. Methods Enzymol. 1986; 122: 251-255Crossref PubMed Scopus (13) Google Scholar). A column (1.5 × 10 cm) was packed with about 8.0 ml of settled gel and equilibrated with 0.01 M potassium phosphate buffer, pH 7.0, containing 10 mM 2-ME and 1 mM NaN3 (buffer 1). Medium (200 ml), plus 2-ME and NaN3 at 10 and 1 mM, respectively, was applied to the affinity column. The column was then washed with buffer 1 (100 ml) followed by the same buffer containing 1 M KCl (100 ml). The enzyme was then eluted from the column with buffer 1 containing 1 M KCl and 20 mM folic acid. The eluate was passed through a column of Bio-Gel P6-DG (Bio-Rad) equilibrated with buffer 1 at pH 6.2 to remove excess folate. The eluate was concentrated to approximately 5 ml using an Amicon (Beverly, MA) filtration cell. Additional purification was done on a DE-52 column with the use of a linear NaCl gradient (0-0.5 M). The enzyme peak was eluted at 0.21 M NaCl, collected, desalted on a column of Bio-Gel P6-DG, and stored at 4°C. The mutant enzyme was purified by the same procedure. Affinity chromatography resulted in purification of both wild type and mutant recombinant protein from most of the proteins of bovine serum. Additional purification on a DE-52 column gave homogeneous preparations for both proteins.Molecular Size DeterminationA molecular size detector (model DynaPro-801, Protein Solutions, Inc., Charlottesville, VA) employing laser-light scattering methodology (18Yarmush D.M. Murphy R.M. Colton C.K. Fisch M. Yarmush M.L. Mol. Immunol. 1988; 25: 17-32Crossref PubMed Scopus (28) Google Scholar, 19Cleland J.L. Wang D.I.C. Biochemistry. 1990; 29: 11072-11078Crossref PubMed Scopus (146) Google Scholar, 20Ponting C.P. Holland S.K. Cederholm-Williams S.A. Marshall J.M. Brown A.J. Spraggon G. Blake C.C.F. Biochim. Biophys. Acta. 1992; 1159: 155-161Crossref PubMed Scopus (42) Google Scholar) was used to analyze recombinant 10-FTHFDH under non-denaturing conditions. The enzyme (in 0.01 M potassium phosphate buffer, pH 7.0, containing 10 mM 2-ME and 1 mM NaN3) was concentrated using a Centricon 30 (Amicon) to about 1 mg/ml. Aliquots of this solution (200 μl) were filtered (0.45 μm; Millipore Co., Bedford, MA) to remove any dust particles prior to measurement. Each sample was measured for 20 cycles and the results analyzed using AutoPro 801 Data Analysis Software (Protein Solutions).Measurement of Enzyme ActivityAll assays were performed at 30°C in a Perkin-Elmer Lambda 4B double beam spectrophotometer (Norwalk, CT). For measurement of hydrolase activity, the reaction mixture contained 0.05 M Tris-HCl, pH 7.8, 100 mM 2-ME, and varying amounts of substrate, either 10-HCO-H4PteGlu or 10-FDDF (0.5-18 μM). The enzyme (1 μg) was added in a final volume of 1.0 ml. The reaction was started by the addition of enzyme and read against a blank cuvette containing all components except enzyme. Appearance of product was measured at either 295 nm for 5,8-dideazafolate or 300 nm for H4PteGlu using molar extinction coefficients of 18.9 × 103 and 21.7 × 103 for 5,8-dideazafolate (21Smith G.K. Mueller W.T. Benkovic P.A. Benkovic S.J. Biochemistry. 1981; 20: 1241-1245Crossref PubMed Scopus (64) Google Scholar) and H4PteGlu(22Case G.L. Kaisaki P.J. Steele R.D. J. Biol. Chem. 1988; 263: 1024-1027Abstract Full Text PDF Google Scholar), respectively. Addition of NADP + to the reaction mixture provided a measure of both dehydrogenase and hydrolase activity, i.e. total activity of the enzyme. Hydrolase activity measured in the absence of NADP + was subtracted from the total activity to give the dehydrogenase activity. Dehydrogenase activity was also measured independently using the increase in absorbance at 340 nm due to production of NADPH and the molar extinction coefficient of 6.2 × 103.Aldehyde dehydrogenase activity was assayed using propanal essentially as described by Lindahl and Evces(23Lindahl R. Evces S. Biochem. Pharmacol. 1984; 33: 3383-3389Crossref PubMed Scopus (68) Google Scholar). The reaction mixture contained 60 mM sodium pyrophosphate buffer, pH 8.5, 5 mM propanal, 1 mM NADP + , and enzyme in a total volume of 1 ml. Activity was estimated from the increase in absorbance at 340 nm.Fluorescence StudiesBinding of NADP + to 10-FTHFDH was detected by measuring the quenching of enzyme fluorescence. These experiments were done on a Perkin-Elmer model 650-40 fluorescence spectrophotometer. Protein samples (9.0 nM) were in 50 mM Tris-HCl, pH 7.8, and 50 mM 2-ME. The NADP + concentration was varied from 0.02 μM to 10 μM. Fluorescence excitation was at 291 nm, and the emission was monitored at 340 nM(24Birdsall B. King R.W. Wheeler M.R. Lewis C.A. Goode S.R. Dunlar R.B. Roberts G.C.K. Anal. Biochem. 1983; 132: 353-361Crossref PubMed Scopus (204) Google Scholar). Kd for NADP + was calculated from data on fluorescence quenching in the presence of NADP + , which were plotted in a linear form(25Chow S.F. Horowitz P.M. J. Biol. Chem. 1985; 260: 15516-15521Abstract Full Text PDF PubMed Google Scholar).RESULTSAnalysis of 10-FTHFDH Oligomeric StructureNative 10-FTHFDH has been reported to exist as a dimer (16Cook R.J. Wagner C. Biochemistry. 1982; 21: 4427-4434Crossref PubMed Scopus (31) Google Scholar) or tetramer(2Rios-Orlandi E.M. Zarkadas C.G. MacKenzie R.E. Biochim. Biophys. Acta. 1986; 871: 24-35Crossref PubMed Scopus (36) Google Scholar, 3Scrutton M.C. Beis I. Biochem. J. 1979; 177: 833-846Crossref PubMed Scopus (28) Google Scholar). Using a light scattering technique, we investigated the oligomeric structure of wild type and mutant recombinant 10-FTHFDH. The analysis showed that both enzymes form monodisperse solutions with a Stokes radius of 7.63 nm and estimated molecular mass of 398.77 kDa for the wild type enzyme and 7.53 nm and 393.15 kDa for the mutant enzyme. This indicates that both wild type and mutant recombinant 10-FTHFDH are tetramers of the 99-kDa monomers(4Cook R.J. Lloyd R.S. Wagner C. J. Biol. Chem. 1991; 266: 4965-4973Abstract Full Text PDF PubMed Google Scholar).Enzymatic Activity of Mutant 10-FTHFDHThe rationale for performing the site-directed mutagenesis was to confirm the putative role of Cys-707 in the catalysis of the dehydrogenase reaction. Assay of the C707A mutant in the presence of NADP showed no dehydrogenase activity with either 10-HCO-H4PteGlu or 10-FDDF (Fig. 2). The wild type recombinant enzyme assayed under the same conditions showed dehydrogenase activity comparable to the native rat liver enzyme.Figure 2:Activity of wild type and mutant 10-FTHFDH. HYD, hydrolase activity; DH, dehydrogenase activity; TOTAL, total activity (measures both dehydrogenase and hydrolase activity). The assays were performed as described under "Experimental Procedures." A, 10-HCO-H4PteGlu used as a substrate; B, 10-FDDF used as a substrate.View Large Image Figure ViewerDownload Hi-res image Download (PPT)It has been shown that 10-FTHFDH is able to oxidize a series of aldehyde substrates in the presence of NADP(4Cook R.J. Lloyd R.S. Wagner C. J. Biol. Chem. 1991; 266: 4965-4973Abstract Full Text PDF PubMed Google Scholar). Kinetic analysis of propanal oxidation by recombinant 10-FTHFDH showed a Km of 638 μM and an estimated Vmax of 245 nmol of NADP min -1 mg -1 . These parameter were similar to those of the natural rat liver enzyme. ( 2R. J. Cook and C. Wagner, unpublished results.) The C707A mutant enzyme was unable to oxidize propanal. These results show that the C707A mutant 10-FTHFDH has completely lost dehydrogenase activity.Analysis of activity of mutant 10-FTHFDH in the absence of NADP + showed that the C707A mutant was able to catalyze the hydrolase reaction. Hydrolase activity of the C707A mutant 10-FTHFDH was comparable to the activity of wild type recombinant enzyme for both 10-HCO-H4PteGlu and 10-FDDF (Table 1).Tabled 1Effect of NADP + on 10-FTHFDH FluorescenceTo check whether the mutation influenced the binding of the coenzyme, we titrated the quenching of tryptophan fluorescence with NADP + . No significant difference in fluorescence quenching between wild type and C707A mutant recombinant 10-FTHFDH was observed (Fig. 3). The Kd for NADP + binding calculated from these data were approximately 0.3 μM for both wild type and mutant enzyme.Figure 3:Fluorescence titration of 10-FTHFDH with NADP + . Curves 1 (opencircles), wild type recombinant 10-FTHFDH; curves2 (darkcircles), C707A mutant 10-FTHFDH. Insertion shows the fluorescence data plotted in linear form. The value (1 - F/Fα ) -1 was plotted against the inverse of NADP + concentration(25Chow S.F. Horowitz P.M. J. Biol. Chem. 1985; 260: 15516-15521Abstract Full Text PDF PubMed Google Scholar). This is a modified form of the classical Stern-Volmer plot, which relates the drop in fluorescence to the concentration of a collisional quencher (see (26Eftink M.R. Ghiron C.A. Anal. Biochem. 1981; 114: 199-227Crossref PubMed Scopus (1613) Google Scholar) for review). F is intrinsic fluorescence observed at an NADP + concentration; Fα is fluorescence in the absence of NADP + . The slope of the line (least squares fit) gave a Kd that was approximately 0.3 μM for both wild type and the C707A mutant 10-FTHFDH. The protein was excited at 291 nm and the emission monitored at 340 nm. 9.0 nM of each enzyme was used for the analysis, and concentration of NADP + was varied from 0.02 μM to 10.0 μM. Variation of the measured values was about 5%.View Large Image Figure ViewerDownload Hi-res image Download (PPT)DISCUSSIONThe sequence identity between 10-FTHFDH and the group of aldehyde dehydrogenases suggested that Cys-707 could be the active site nucleophile. In order to test this hypothesis, Cys-707 in 10-FTHFDH was modified. The closest amino acid substitution for cysteine is serine. Serine can, however, function as a nucleophile in place of cysteine in some enzyme-catalyzed reactions(27Witkowski A. Witkowska H.E. Smith S. J. Biol. Chem. 1994; 269: 379-383Abstract Full Text PDF PubMed Google Scholar). Therefore we replaced Cys-707 with alanine. Such a substitution is used often in site-directed mutagenesis to study the role of cysteine residues. In our experiments mutation of Cys-707 of 10-FTHFDH to Ala resulted in complete loss of dehydrogenase activity of the enzyme for both the native substrate 10-HCO-H4PteGlu and 10-FDDF. The C707A mutant also lost the ability to oxidize propanal, while wild type recombinant 10-FTHFDH oxidized propanal as well as the native rat liver enzyme. Measurement of binding of the coenzyme, NADP + , by fluorescence titration showed no difference between the mutant and wild type enzyme, indicating that loss of dehydrogenase activity was not due to any change in the coenzyme binding site.Substitution of a single amino acid residue can sometimes result in a decrease of enzyme activity even if this residue is not involved in the active site due to changes in the higher order protein structure(28Ackers G.K. Smith F.R. Annu. Rev. Biochem. 1985; 54: 597-629Crossref PubMed Scopus (197) Google Scholar). Such critical residues play a basic role in protein folding and/or in supporting correct protein structure(29Matthews B.W. Biochemistry. 1987; 26: 6885-6888Crossref PubMed Scopus (185) Google Scholar, 30Goldenberg D.P. Creighton T.E. Protein Folding. W. H. Freeman, New York1992: 353-403Google Scholar). Replacement of such residues could lead to decreased protein stability (31Garvey E.P. Matthews C.R. Narang S.A. Protein Engineering. Butterworths, Boston1990: 37-63Crossref Google Scholar) and also to a decreased level of expression due to higher accessibility of mutant proteins to proteases(32Hanioka N. Gonzalez F.J. Lindberg G.L. Korzekwa K.R. J. Steroid Biochem. Mol. Biol. 1992; 43: 1037-1043Crossref PubMed Scopus (5) Google Scholar). On the other hand, many amino acid substitutions do not have large effects on protein stability(33Pakula A.A. Sauer R.T. Nature. 1990; 344: 363-364Crossref PubMed Scopus (150) Google Scholar, 34Alber T. Annu. Rev. Biochem. 1989; 58: 765-798Crossref PubMed Google Scholar). Even when a replaced residue is essential for protein conformation, protein structures adjust to compensate for changes in sequence(29Matthews B.W. Biochemistry. 1987; 26: 6885-6888Crossref PubMed Scopus (185) Google Scholar). Our study did not reveal any changes in stability of the C707A mutant in comparison with the wild type protein and both displayed a similar level of expression. Moreover, the Kd values for NADP + were similar for both enzymes. The behavior of the mutant during the purification procedure was also identical to that of the wild type recombinant protein, indicating no gross alteration of the native structure(33Pakula A.A. Sauer R.T. Nature. 1990; 344: 363-364Crossref PubMed Scopus (150) Google Scholar). It is known that native 10-FTHFDH forms oligomers(2Rios-Orlandi E.M. Zarkadas C.G. MacKenzie R.E. Biochim. Biophys. Acta. 1986; 871: 24-35Crossref PubMed Scopus (36) Google Scholar, 3Scrutton M.C. Beis I. Biochem. J. 1979; 177: 833-846Crossref PubMed Scopus (28) Google Scholar, 16Cook R.J. Wagner C. Biochemistry. 1982; 21: 4427-4434Crossref PubMed Scopus (31) Google Scholar); therefore, we also determined the oligomeric structure of the wild type recombinant and the mutant enzymes. For this purpose, we used a laser-light scattering technique. This technique involves analysis of the temporal fluctuations in the intensity of light scattered by the Brownian motion of macromolecules. It provides a rapid, accurate, and noninvasive method of determining the translational diffusion coefficient of macromolecules that can be related to an effective size(18Yarmush D.M. Murphy R.M. Colton C.K. Fisch M. Yarmush M.L. Mol. Immunol. 1988; 25: 17-32Crossref PubMed Scopus (28) Google Scholar). The analysis revealed that both wild type and mutant proteins existed as tetramers. The fact that the entire population of the active wild type recombinant 10-FTHFDH was organized as a tetramer indicates that this is the functional form of the enzyme. Since the C707A mutant 10-FTHFDH also forms a tetramer, this suggests they have similar conformations. Based on all these observations, we assume that the substitution of an alanine for the cysteine did not change protein conformation and stability. However, conservative mutations that dramatically reduce activity without changes in protein stability strongly suggest that a residue is important for substrate recognition or activity.The mutation of Cys-707 to Ala did not produce any changes in the hydrolase activity of 10-FTHFDH. This indicates that the enzyme has two different catalytic sites. This observation is not surprising since Rios-Orlandi et al.(2Rios-Orlandi E.M. Zarkadas C.G. MacKenzie R.E. Biochim. Biophys. Acta. 1986; 871: 24-35Crossref PubMed Scopus (36) Google Scholar) showed that both dehydrogenase and hydrolase activities can take place simultaneously. The existence of two different functional domains was predicted when the sequence of the enzyme was determined(4Cook R.J. Lloyd R.S. Wagner C. J. Biol. Chem. 1991; 266: 4965-4973Abstract Full Text PDF PubMed Google Scholar). A recent study by Schirch et al.(35Schirch D. Villar E. Maras B. Barra D. Schirch V. J. Biol. Chem. 1994; 269: 24728-24735Abstract Full Text PDF PubMed Google Scholar) where differential scanning calorimetry and proteolytic digestion were used has also shown the hydrolase and dehydrogenase activities of rabbit liver 10-FTHFDH reside in different domains.The results show that cysteine 707 is a key residue of the dehydrogenase active site of 10-FTHFDH. Similar results were obtained for aldehyde dehydrogenase from rat liver mitochondria(6Weiner H. Farres J. Wang T.T.Y. Cunningham S.J. Zheng C.-F. Ghenbot G. Weiner H. Wermuth B. Crabb D.W. Enzymology and Molecular Biology of Carbonyl Metabolism. Vol. 3. Plenum Press, New York1991: 13-17Google Scholar), which has 46% homology with the carboxyl-terminal domain of rat 10-FTHFDH(4Cook R.J. Lloyd R.S. Wagner C. J. Biol. Chem. 1991; 266: 4965-4973Abstract Full Text PDF PubMed Google Scholar). Aldehyde dehydrogenase with the corresponding C302A mutation was devoid of activity(6Weiner H. Farres J. Wang T.T.Y. Cunningham S.J. Zheng C.-F. Ghenbot G. Weiner H. Wermuth B. Crabb D.W. Enzymology and Molecular Biology of Carbonyl Metabolism. Vol. 3. Plenum Press, New York1991: 13-17Google Scholar). At the same time substitution an alanine for 2 other cysteines in positions 49 and 162 of aldehyde dehydrogenase did not result in changes of enzyme activity(6Weiner H. Farres J. Wang T.T.Y. Cunningham S.J. Zheng C.-F. Ghenbot G. Weiner H. Wermuth B. Crabb D.W. Enzymology and Molecular Biology of Carbonyl Metabolism. Vol. 3. Plenum Press, New York1991: 13-17Google Scholar). Apparently, the dehydrogenase catalytic site of 10-FTHFDH is structurally similar to that of aldehyde dehydrogenase with a cysteine as a catalytic nucleophile residue and supports the role of a thiohemiacetal intermediate in the reaction mechanism. INTRODUCTION10-Formyltetrahydrofolate dehydrogenase (10-FTHFDH) ( 1The abbreviations used are: 10-FTHFDH10-formyltetrahydrofolate dehydrogenase10-HCO-H4PteGlu10-formyltetrahydrofolateH4PteGlutetrahydrofolate10-FDDF10-formyl-5,8-dideazafolate2-ME2-mercaptoethanolPCRpolymerase chain reaction.) (EC 1.5.1.6) catalyzes both the NADP + -dependent oxidation of 10-formyltetrahydrofolate (10-HCO-H4PteGlu) to tetrahydrofolate and CO2 and the NADP + -independent hydrolysis of 10-HCO-H4PteGlu to tetrahydrofolate and formate(1Kutzbach C. Stokstad E.L.R. Methods Enzymol. 1971; 18B: 793-798Crossref Scopus (29) Google Scholar, 2Rios-Orlandi E.M. Zarkadas C.G. MacKenzie R.E. Biochim. Biophys. Acta. 1986; 871: 24-35Crossref PubMed Scopus (36) Google Scholar, 3Scrutton M.C. Beis I. Biochem. J. 1979; 177: 833-846Crossref PubMed Scopus (28) Google Scholar). The amino-terminal domain of rat liver 10-FTHFDH (residues 1-203) is 24-30% identical to a group of glycinamide ribonucleotide transformylases (EC 2.1.2.1) from different species(4Cook R.J. Lloyd R.S. Wagner C. J. Biol. Chem. 1991; 266: 4965-4973Abstract Full Text PDF PubMed Google Scholar). The carboxyl-terminal domain (residues 417-902) of 10-FTHFDH has 46% identity with a series of NAD + -dependent aldehyde dehydrogenases (EC 1.2.1.3), and the enzyme is able to oxidize aldehydes(4Cook R.J. Lloyd R.S. Wagner C. J. Biol. Chem. 1991; 266: 4965-4973Abstract Full Text PDF PubMed Google Scholar).All known aldehyde dehydrogenases contain a conserved cysteine, which, in the case of 10-FTHFDH, is Cys-707(4Cook R.J. Lloyd R.S. Wagner C. J. Biol. Chem. 1991; 266: 4965-4973Abstract Full Text PDF PubMed Google Scholar, 5Hempel J. Nicholas H. Lindahl R. Protein Sci. 1993; 2: 1890-1900Crossref PubMed Scopus (166) Google Scholar). The conserved cysteine is presumed to act as a nucleophile in the formation of an enzyme-linked thiohemiacetal intermediate(6Weiner H. Farres J. Wang T.T.Y. Cunningham S.J. Zheng C.-F. Ghenbot G. Weiner H. Wermuth B. Crabb D.W. Enzymology and Molecular Biology of Carbonyl Metabolism. Vol. 3. Plenum Press, New York1991: 13-17Google Scholar, 7Blatter E.E. Abriola D.P. Pietruszko R. Biochem. J. 1992; 282: 353-360Crossref PubMed Scopus (39) Google Scholar). A number of different approaches may be used to identify the amino acid at the active site of an enzyme. Using an affinity label, Blatter et al.(7Blatter E.E. Abriola D.P. Pietruszko R. Biochem. J. 1992; 282: 353-360Crossref PubMed Scopus (39) Google Scholar) found that Cys-302 formed a covalent intermediate with the substrate in human aldehyde dehydrogenase(7Blatter E.E. Abriola D.P. Pietruszko R. Biochem. J. 1992; 282: 353-360Crossref PubMed Scopus (39) Google Scholar). Site-directed mutagenesis confirmed that Cys-302 is essential for enzyme activity of rat liver mitochondrial aldehyde dehydrogenase, whereas Cys-49 and Cys-162 can be changed to alanine without altering enzyme activity(6Weiner H. Farres J. Wang T.T.Y. Cunningham S.J. Zheng C.-F. Ghenbot G. Weiner H. Wermuth B. Crabb D.W. Enzymology and Molecular Biology of Carbonyl Metabolism. Vol. 3. Plenum Press, New York1991: 13-17Google Scholar). Additionally, site-directed mutagenesis experiments have shown that serine 74 is also important for enzyme activity(6Weiner H. Farres J. Wang T.T.Y. Cunningham S.J. Zheng C.-F. Ghenbot G. Weiner H. Wermuth B. Crabb D.W. Enzymology and Molecular Biology of Carbonyl Metabolism. Vol. 3. Plenum Press, New York1991: 13-17Google Scholar). This observation was unexpected because this serine is conserved only in mammalian aldehyde dehydrogenases(5Hempel J. Nicholas H. Lindahl R. Protein Sci. 1993; 2: 1890-1900Crossref PubMed Scopus (166) Google Scholar). It has been shown recently that serine 74 is not involved in aldehyde oxidation but may be involved in NAD + binding(8Rout U.K. Weiner H. Biochemistry. 1994; 33: 8955-8961Crossref PubMed Scopus (17) Google Scholar). In the present study we expressed rat 10-FTHFDH in a baculovirus system and used site-directed mutagenesis to elucidate the role of the highly conserved Cys-707 in the activity of the enzyme.
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