7356
Biochemistry 2004, 43, 7356-7364
Roles for Cysteine Residues in the Regulatory CXXC Motif of Human Mitochondrial Branched Chain Aminotransferase Enzyme† Myra E. Conway, Leslie B. Poole, and Susan M. Hutson* Department of Biochemistry, Wake Forest UniVersity School of Medicine, Medical Center BouleVard, Winston-Salem, North Carolina 27157 ReceiVed January 27, 2004; ReVised Manuscript ReceiVed March 29, 2004
ABSTRACT: The redox-active dithiol/disulfide C315-Xaa-Xaa-C318 center has been implicated in the regulation of the human mitochondrial branched chain aminotransferase isozyme (hBCATm) [Conway, M. E., Yennawar, N., Wallin, R., Poole, L. B., and Hutson, S. M. (2002) Biochemistry 41, 9070-9078]. To explore further the mechanistic details of this CXXC center, mutants of the Cys residues at positions 315 and 318 of hBCATm were individually and in combination converted to alanine or serine by sitedirected mutagenesis (C315A, C315S, C318A, C318S, C315/318A, and C315/318S). The effects of these mutations on cofactor absorbance, secondary structures, steady-state kinetics, and sensitivity toward hydrogen peroxide (H2O2) treatment were examined. Neither the UV-visible spectroscopic studies nor the circular dichroism data showed any major perturbations in the structure of the mutants. Kinetic analyses of the CXXC mutant proteins indicated primarily a modest reduction in kcat with no significant change in Km. The largest effect on the steady-state kinetics was observed with the C315 single mutants, in which substitution of the thiol group resulted in a reduced kcat (to 26-33% of that of wild-type hBCATm). Moreover, the C315 single mutants lost their sensitivity to oxidation by H2O2. The kinetic parameters of the C318 mutants were largely unaffected by the substitutions, and as with wild-type hBCATm, reaction of the C318A mutant protein with H2O2 resulted in the complete loss of activity. In the case of oxidized C318A, this loss was largely irreversible on incubation with dithiothreitol. Mass spectrometry and dimedone modification results revealed overoxidation of the thiol group at position 315 to sulfonic acid occurring via a sulfenic acid intermediate in the H2O2-treated C318A enzyme. Thus, C315 appears to be the sensor for redox regulation of BCAT activity, whereas C318 acts as the “resolving cysteine”, allowing for reversible formation of a disulfide bond.
Transamination of the branched chain amino acids (BCAA),1 leucine, isoleucine, and valine, to their respective R-keto acids, R-ketoisocaproate, R-keto-β-methylvalerate, and R-ketoisovalerate, respectively, is catalyzed by pyridoxal phosphate (PLP)-dependent branched chain aminotransferases (BCATs) (EC 2.6.1.42). There are two BCAT isozymes in humans, a mitochondrial (hBCATm) form found † This study was supported by a grant from the National Institutes of Health to S.M.H. (RO1 DK34738) and an Established Investigatorship from the American Heart Association to L.B.P. (0140175N). The spectrometer with the electrospray source was purchased with funding from the National Science Foundation (BIR-9414018), the North Carolina Biotechnology Center (9903IDG-1002), and the WinstonSalem Foundation. Partial support for the Analytical Chemistry Laboratory came from the Comprehensive Cancer Center of Wake Forest University (National Cancer Institute Center Grant CA12107). * To whom correspondence should be addressed: Department of Biochemistry, Wake Forest University School of Medicine, Medical Center Boulevard, Winston-Salem, NC 27157. Telephone: (336) 7137217. Fax: (336) 716-7671. E-mail:
[email protected]. 1 Abbreviations: BCAA, branched chain amino acids; BCAT, branched chain aminotransferase; hBCATm, human mitochondrial branched chain aminotransferase; hBCATc, human cytosolic branched chain aminotransferase; WT, wild-type; CXXC mutant proteins, C315A, C318A, C315/318A, C315S, C318S, and C315/318S; DTNB, 5,5′dithiobis(2-nitrobenzoic acid); DTT, dithiothreitol; PLP, pyridoxal phosphate; HEPES, N-(2-hydroxyethyl)piperazine-N′-2-ethanesulfonic acid; H2O2, hydrogen peroxide; ESI-MS, electrospray ionization mass spectrometry; TNB, 2-nitro-5-thiobenzoate; NBD chloride, 7-chloro4-nitrobenz-2-oxa-1,3-diazole; NBD, 4-nitrobenz-2-oxa-1,3-diazole.
in most tissues and a cytosolic (hBCATc) form which is localized primarily in the central nervous system (for a review, see ref 1). Furthermore, two alternatively spliced isoforms of hBCATm have been reported recently. One contains a 12-amino acid deletion that starts immediately following C315. This isoform binds to and acts as a corepressor of thyroid hormone receptor β1 (2). The second isoform contains a 100-amino acid deletion that starts shortly after the N-terminal methionine. Thus, it lacks the mitochondrial targeting sequence, and is found in the cytosol of placenta and most human tissues (3). The BCATs are classified in the fold type IV class of PLPdependent enzymes (4-7). The members of this class include the BCAT enzymes as well as bacterial D-amino acid aminotransferase (which has stereospecificity that is the opposite of the BCAT, D-amino acids vs L-amino acids) (5), and bacterial 4-amino-4-deoxychorismate lyase (8). A unique feature of this group of three enzymes is that the proton is added to or abstracted from the C4′ atom of the coenzymeimine or external aldimine intermediate on the re face instead of the si face of the PLP cofactor (9). A distinctive feature of hBCATm and hBCATc proteins is a CXXC motif located ∼10 Å from the active site (7). Multiple-sequence alignments of the BCAT proteins indicate that this CXXC motif is conserved in mammalian proteins but not in lower eukaryotes or prokaryotes or in other fold
10.1021/bi0498050 CCC: $27.50 © 2004 American Chemical Society Published on Web 05/18/2004
Regulatory Mechanism of the CXXC Motif in hBCATm type IV PLP enzymes (10). X-ray crystallographic results indicate that the short distance between the sulfur atoms (3.09-3.46 Å) of the two CXXC cysteines is compatible with disulfide bond formation under oxidizing conditions (7). Recently, it was shown that modification of C315 and C318 with thiol specific reagents or oxidation of the thiols to a disulfide bond results in the loss of hBCATm activity (10). Moreover, addition of dithiothreitol (DTT) completely reversed the oxidation and restored activity, suggesting that this peroxide-sensitive CXXC center is involved in the redoxlinked regulation of hBCATm activity (10). To further understand the regulation of hBCATm by this redox-active CXXC motif, either C315, C318, or both were mutated to alanine or serine using site-directed mutagenesis. As reported herein, the results strongly suggest that C315 is the peroxide-reactive thiol(ate) functioning as the redox sensor in the regulation of hBCATm. The second cysteine, C318, subsequently reacts with the nascent sulfenic acid form of C315, forming a disulfide bond and permitting reversible regulation by preventing overoxidation of hBCATm. EXPERIMENTAL PROCEDURES Materials. DTT, 5,5′-dithiobis(2-nitrobenzoic acid) (DTNB), dimedone, R-ketoisocaproate, R-ketoisovalerate, and PLP were obtained from Sigma (St. Louis, MO). Hydrogen peroxide (H2O2) (30%) was purchased from Fisher Scientific (Suwanee, GA). The PD10 columns and the Mono-Q HR 5/5 (1 mL) anion-exchange column were from Amersham Pharmacia Biotech (Piscataway, NJ). Apollo ultrafiltration devices (7 mL) were from Orbital Biosciences (Topsfield, MA). The QuikChange site-directed mutagenesis kit was from Stratagene Corp. (La Jolla, CA). The QIAprep spin miniprep kit and the MinEluate gel extraction kit were from Qiagen (Valencia, CA). Oligonucleotides were synthesized by MWG Biotech (High Point, NC). Purified human thrombin was obtained from Enzyme Research Laboratories (South Bend, IN). Site-Directed Mutagenesis of Wild-Type (WT) hBCATm. The hBCATm cDNA clone previously ligated into the pET28a expression vector (11) was used as the plasmid DNA template for oligonucleotide-directed mutagenesis in this study. To substitute C315 and C318 with alanine or serine in either position or both, synthetic oligonucleotides and their exact complements containing the desired mutations (C315A, C318A, C315/318A, C315S, C318S, C315/318S, and C108S) were designed. The following sense primers were used: 5′TCG GGC ACC GCT GCG CAG GTC TGC CCA-3′ for C315A, 5′-GCT TGC CAG GTC GCG CCA GTG CAC CGA-3′ for C318A, 5′-TCG GGC ACC GCT TCC CAG GTC TGC CCA-3′ for C315S, 5′-GCT TGC CAG GTC TCC CCA GTG CAC CGA-3′ for C318S, 5′-GGC ACC GCT GCG CAG GTC GCG CCA GTG CAC CGA-3′ for C315/318A, 5′-GGC ACC GCT TCC CAG GTC TCC CCA GTG CAC CGA-3′ for C315/318S, and 5′-GCC ATG CGC CTG TCC CTG CCG AGT TTC-3′ for C108S. The mutagenesis method followed that recommended with the QuikChange kit. Briefly, the PCR was carried out in the MiniCycler PCR machine (MJ Research, Inc.) using the purified double-stranded hBCATm plasmid DNA, and each mutagenic primer pair, for 16 cycles. The PCR product was incubated with DpnI endonuclease, which digests the parental methylated DNA template. Plasmid DNA was purified by agarose gel electrophoresis and extracted using the MinEluate
Biochemistry, Vol. 43, No. 23, 2004 7357 gel extraction kit from Qiagen. The mutated plasmid was transformed into the Epicurian Coli XL1-Blue supercompetent cells, and positive mutant colonies were selected by kanamycin resistance. Colonies were subcultured, and the plasmid DNA was isolated using the QIAprep Spin Miniprep Kit. The desired mutation and fidelity of PCR amplification were confirmed by DNA sequence analysis using the ABI 377 DNA sequenator in the DNA Sequencing Core Laboratory of the Comprehensive Cancer Center of the Wake Forest University School of Medicine. The WT hBCATm and CXXC mutant plasmids were transformed into BL21(DE3) cells as described by Davoodi et al. (11). Expression and Purification of WT hBCATm and Mutant Proteins. Purification of WT and CXXC mutant hBCATm proteins was carried out as previously described by Conway and Hutson (12), with the following modifications. Briefly, hBCATm was extracted from pelleted bacteria using sonication. The histidine-tagged fusion protein was purified using nickel-NTA resin (Qiagen, Chatsworth, CA), followed by digestion with thrombin (100 NIH units) to remove the affinity tag. Purified WT or mutant proteins were obtained after anion-exchange chromatography using the Mono-Q HR 5/5 anion-exchange column (Pharmacia), omitting the hydrophobic interaction chromatography step used by Davoodi et al. (11). The proteins were selectively eluted using a sodium chloride gradient from 0 to 0.5 M in 10 mM potassium phosphate (pH 8.0) over the course of 20 min at a flow rate of 1.0 mL/min. The purified proteins were then dialyzed at 4 °C into a buffer containing 25 mM Tris-HCl (pH 7.5), 150 mM NaCl, 1 mM glucose, 1 mM EDTA, 1 mM R-ketoisocaproate, 5 mM DTT, and approximately 15% glycerol. The WT hBCATm enzyme could be stored at 4 °C for 2 days or at -20 °C for 1.5-2 months. The CXXC mutant proteins were stable at -20 °C for 6 months. The concentration of the purified protein was estimated using the method of Schaffner and Weissmann (13) or determined from the absorbance at 280 nm using the extinction coefficient of 67 600 M-1 cm-1 per monomer (11). The final yield of the pure protein was approximately 10 mg of purified protein/L of Escherichia coli. BCAT Assay and Steady-State Kinetics. The standard assay for BCAT activity was performed at 37 °C by assessing the formation of [1-14C]valine from R-keto[1-14C]isovalerate as described previously (11). The standard assay solution (0.5 mL) contained 25 mM potassium phosphate buffer (pH 7.8), 5 mM DTT, 1 mM R-keto[1-14C]isovalerate, and 12 mM isoleucine at 37 °C as described previously (11, 12). A unit of enzyme activity was defined as 1 µmol of valine formed per minute under standard conditions. All assays were performed in duplicate or triplicate. Specific activities of WT BCATm and the mutant enzymes for different amino acids (including the branched chain amino acids, glutamate, tyrosine, tryptophan, phenylalanine, aspartate, threonine, aspartate, norleucine, and norvaline) were compared using 1 mM R-keto[1-14C]isovalerate and an amino acid concentration of 10 mM in the standard assay buffer. For steady-state kinetic determinations, reaction rates with the amino acid/ R-keto[1-14C]isovalerate pairs were determined holding the concentration of R-ketoisovalerate constant at 2 mM, which was saturating for the BCAA and glutamate. The concentrations of the amino acids isoleucine (0-2 mM), leucine (0-2 mM), valine (0-30 mM), and glutamate (0-40 mM) were varied. Stock solutions of the dicarboxylic acid amino acid
7358 Biochemistry, Vol. 43, No. 23, 2004 glutamate were neutralized with KOH. Data were collected for eight or ten concentrations of each amino acid. Km and Vmax values were calculated from the respective LineweaverBurk plots. Spectrophotometric Measurements. Absorption spectra were obtained with a Beckman DU640 spectrophotometer at a protein concentration of 1 mg/mL for all proteins. Circular dichroism (CD) measurements were determined with a JASCO J-720 spectropolarimeter equipped with a variabletemperature accessory. WT hBCATm and the CXXC mutant proteins were dialyzed into 10 mM potassium phosphate buffer (pH 7.5) containing either 12 mM leucine or 6 mM isoleucine. Excess substrate was then removed by further dialysis in 10 mM potassium phosphate buffer (pH 7.5). CD spectra in the near-UV region were measured in a 1 cm quartz cylindrical cuvette at a protein concentration of 1 mg/mL. In the far-UV region, CD spectra were acquired using a protein concentration of 0.5 mg/mL in 10 mM potassium phosphate buffer and a cuvette with a path length of 0.05 cm. Spectrophotometric Analysis of Thiol Groups. For titration of the solvent-accessible thiol groups in reduced or oxidized hBCATm proteins, 10 nmol of protein was exchanged into buffer containing 50 mM HEPES (pH 7.0) and 1 mM EDTA (buffer A) using a PD10 column (Amersham Biosciences) as described by the manufacturer (12). Two nanomoles of protein were then incubated with a 100-fold excess of DTNB at room temperature for 10 min. The absorbance change at 412 nm was monitored, and the concentration of free thiol groups was calculated from the liberated 2-nitro-5-thiobenzoate (thiolate) dianion (TNB) using a molar extinction coefficient of 14 150 M-1 cm-1 (14). H2O2 SensitiVity of WT hBCATm and the CXXC Mutant Proteins. Proteins were first exchanged into buffer A, and then 7 nmol of protein per aliquot was incubated with varying amounts of H2O2 for 18-24 h at 4 °C, after which aliquots were removed for BCAT activity and thiol content measurements (see above). The remainder of the fraction was incubated with a 100-fold molar excess of DTT for 18 h at 4 °C, after which the activity was again measured as described above. Control samples were incubated under the same conditions without the addition of H2O2. Mass Spectrometry Analysis of WT and CXXC Mutant hBCATm Proteins. The amino acid substitution(s) in each of the mutated proteins was confirmed by mass spectrometry (10) on a Micromass Quattro II triple-quadrupole mass spectrometer (Micromass, Manchester, England) fitted with an electrospray source. Sample pretreatment followed the method previously described by Conway et al. (10). Electrospray ionization mass spectrometry (ESI-MS) analysis of H2O2- and/or dimedone-treated proteins was also employed for sulfenic acid identification and assessment of oxidation rates. Each mutant or WT protein was initially exchanged into buffer A using a PD-10 column and then concentrated using a 7 mL Apollo ultrafiltration device with a 30 kDa cutoff (Orbital Biosciences, Topsfield, MA). Samples (4.5 nmol each) were brought to 210 µL total with additional HEPES buffer, 10 µL of dimethyl sulfoxide, or dimedone in dimethyl sulfoxide (final dimedone concentration of 5 mM), and H2O2 at 0, 1, 1.5, or 2 mM. Following incubation at 25 °C for up to 23 h, catalase (5 µL of a 1 mg/mL stock solution) was added followed by incubation for a further 20 min, and then 15 µL samples were frozen
Conway et al. Table 1: Physical Characterization of the CXXC Mutant Proteins Compared with WT hBCATm
protein
predicted molecular mass (amu)
observed molecular mass (amu)a
moles of thiol groups per mole of proteinb
WT hBCATm C315A C315S C318A C318S C315/318A C315/318S
41 732 41 702 41 717 41 702 41 717 41 672 41 702
41 730 ( 2 41 700 ( 3 41 716 ( 3 41 700 ( 2 41 717 ( 3 41 670 ( 4 41 700 ( 2
2.44 ( 0.03 0.77 ( 0.03 1.05 ( 0.03 1.11 ( 0.02 1.13 ( 0.02 0.44 ( 0.02 0.43 ( 0.01
a Results from mass spectrometry are represented as the mean atomic mass units ( standard errors of the mean. Flow injection analysis was used with the carrier solvent consisting of a 50:50 acetonitrile/water mixture with 1% formic acid. Approximately 10% acetonitrile was added to the protein samples, which were in 10 mM ammonium bicarbonate, and 5-10 µL (1 nmol of WT hBCATm or CXXC mutant protein) of the sample solution was injected. Each sample required 8-20 scans (3 s scans for 1 min), and the data were processed using MassLynx version 3.5 and the Maximum Entropy software supplied with the program to generate spectra on the absolute molecular weight scale. b The moles of thiol groups per mole of protein are reported as the means ( standard errors of the mean from four to six determinations. This was estimated by titrating 2 nmol of protein with a 100fold excess of DTNB and measuring the increase in absorbance at 412 nm over the course of 10 min. The moles of thiol group per mole of protein were determined using the molar extinction coefficient 14 150 M-1 cm-1 for TNB.
on dry ice and stored at -80 °C until they were assayed (within 24 h). The remainder of each sample was exchanged into 10 mM ammonium bicarbonate buffer using four rounds of dilution (to 6.5 mL) and reconcentration (to ∼50 µL) with the Apollo ultrafiltration devices. Particulate matter was removed by centrifugation, and samples were analyzed by ESI-MS after addition of acetonitrile to a final concentration of 50% and formic acid to 1%. RESULTS Expression and Purification of the CXXC Mutant Proteins. Nucleotide sequence analysis showed that the mutations had been introduced in all of the mutant plasmid DNAs and that the sequences of the coding regions were correct. Wild-type hBCATm and CXXC mutant proteins were overexpressed in E. coli strain BL21(DE3) and yielded similar amounts of purified proteins (8-10 mg/L). There were no changes required for the purification of the CXXC mutant proteins compared to the protocol used for WT hBCATm. The purified enzymes migrated as single bands with an approximate molecular mass of 42 kDa on SDS-PAGE, and the purity of each protein was judged to be >98% (data not shown). ESI-MS showed that the observed molecular mass of the respective mutant enzymes corresponded to the predicted molecular masses (Table 1). A previous study indicated that C315 and C318 represent the two most reactive thiols in reduced hBCATm (10). To evaluate whether mutagenesis of one or both of the cysteine residues in the CXXC motif resulted in the concomitant loss of the predicted number of free thiols in the mutant proteins, DTNB titrations were performed on all proteins. As shown in Table 1 and in agreement with previous reports (10, 11), approximately two thiol groups per monomer were titrated in reduced, native WT hBCATm. For the C315A, C318A, C315S, and C318S hBCATm mutants, approximately one
Regulatory Mechanism of the CXXC Motif in hBCATm
FIGURE 1: Titration of WT hBCATm and the CXXC mutant proteins with DTNB. Two micromoles of protein was incubated with a 100-fold excess of DTNB, and the increase in absorbance was measured at 412 nm over the course of 20 min: WT hBCATm (b), C108S (O), C318S (9), C315S (2), and C315,318S (0). The line at 600 s indicates the point at which values were taken for Table 1.
thiol reacted with DTNB within 10 min, whereas with the C315/318A and C315/318S mutants,
