Báo cáo khoa học: Brain succinic semialdehyde dehydrogenase Reactions of sulfhydryl residues connected with catalytic activity pdf

Brain succinic semialdehyde dehydrogenase Reactions of sulfhydryl residues connected with catalytic activity Byung Ryong Lee 1 , Dae Won Kim 1 , Joung-Woo Hong 2 , Won Sik Eum 1 , Hee Soon Choi 1 , Soo Hyun Choi 1 , So Young Kim 1 , Jae Jin An 1 , Jee-Yin Ahn 1, *, Oh-Shin Kwon 3 , Tae-Cheon Kang 4 , Moo Ho Won 4 , Sung-Woo Cho 5 , Kil Soo Lee 1 , Jinseu Park 1 and Soo Young Choi 1 1 Department of Genetic Engineering and Research Institute for Bioscience and Biotechnology, Hallym University, Chunchon, Korea; 2 Department of Molecular and Cellular Biochemistry, The Ohio State University, Columbus, OH, USA; 3 Department of Biochemistry, Kyungpook National University, Taegu, Korea; 4 Department of Anatomy, College of Medicine, Hallym University, Chunchon, Korea; 5 Department of Biochemistry and Molecular Biology, University of Ulsan College of Medicine, Seoul, Korea Incubation of an NAD + -dependent succinic semialdehyde dehydrogenase f rom bovine brain with 4-dimethylamino- azobenzene-4-iodoacetamide (DABIA) resulted in a time- dependent loss of enzymatic activity. This inactivation followed pseudo fir st-order kinetics with a second-order rate constant of 168 M )1 Æmin )1 . The spectrum of DABIA-labeled enzyme show ed a characteristic p eak of t he DABIA a lkyl- ated sulfhydryl group chromophore at 436 nm, which was absent from the spectrum of the native enzyme. A linear relationship was observed between DABIA binding and t he loss of enzyme activity, which extrapolates to a stoichio- metry of 8.0 mol DABIA derivatives per mol enzyme tetramer. This inactivation was prevented by p reincubating the enzyme with substrate, succinic semialdehyde, but not b y preincubating with coenzyme NAD + . After tryptic diges- tion of the enzyme modified with DABIA, two peptides absorbing a t 436 nm were isolated by reverse-phase HPLC. The amino acid sequences of the DABIA-labeled pep- tides were VCSNQFLVQR and EVGEAICTDPLVSK, respectively. These s ites are identical to the putative active site sequences of other brain succinic semialdehyde dehy- drogenases. These results suggest that the c atalytic function of succinic s emialdehyde dehydrogenase is inhibited by the specific binding of DABIA to a cysteine residue at or near its active site. Keywords: brain succinic semialdehyde d ehydrogenase; DABIA; GABA shunt; reactive cysteine residues. c-Aminobutyric acid (GABA) is produced from glutamate in a reaction catalyzed by glutamate decarboxylase (GAD) and further metabolized to succinate by the successive action of GABA transaminase (GABA-T) and succinic semialdehyde dehydrogenase (SSADH). The carbon skel- etal of GABA therefore enters the tricarboxylic acid in the form of succinate. GABA metabolism has been well characterized in the mammalian central nervous system where GABA functions as a major inhibitory n eurotrans- mitter. SSADH, the final enzyme in GABA metabolism, has been purified from rat, human an d bovine brain [1–3]. T his enzyme is also the site of an inborn error of human metabolism [4]. In a utosomal recessively inherited SSADH deficiency, now identified in more than 45 patients who manifest varying d egrees of psychomotor retardation with speech delay, the normal o xidative pathway is blocked, thereby resulting in the a ccumulation of succinic semialde- hyde (SSA). Metabolic patterns in physiologic fluids derived from patients show large increases in gamma-hydroxybu- tyrate (GHB) [5], t he reduction p roduct of SSA by succinic semialdehyde reductase [6]. GHB, the biochemical hallmark of SSADH deficiency, produces central n ervous system effects including altered motor activity and behavior disturbances when administered to animals and humans at pharmacologic levels [7]. Recently, an SSADH cDNA was c loned from rat brain and human liver [8]. The mammalian SSADH bears signi- ficant homology to bacterial NADP + -SSADH and con- served regions of aldehyde dehydrogenases, suggesting that it is a member of the aldehyde dehydrogenase superfamily. SSADH cD NA and g enomic sequences have been used to identify two point mutations in the SSADH genes derived from fo ur patients [9]. Splicing mutations resulted in exon skipping in all four cases. In addition, a frameshift a nd pre- mature termination was observed in one case and an in-frame deletion in the resulting protein was detected in the other case. Parents an d s iblings were shown to b e Correspondence to S. Y. Choi, Department of Genetic Engineering, Hallym, University, Chunchon 200-702, Korea. Fax: +82 33 241 1463, Tel.: +82 33 248 2112, E-mail: sychoi@hallym.ac.kr or O S. Kwon, Department of Biochemistry, Kyungpook National University, Taegu 702-701, Korea. Tel.: +82 53 950 6356, E- mail: oskwon@knu.ac.kr Abbreviations: DABIA, 4-dimethylaminoazobenzene-4-iodoaceta- mide; Dys, DABIA-Cys; GAD, glutamate decarboxylase; GABA, c-aminobutyric acid; GABA-T, c-aminobutyric acid transaminase; GBH, gamma-hydroxybutyrate; SSA, succinic semialdehyde; SSADH, succinic semialdehyde dehydr ogenase. *Present address: Department o f Pathology, S chool of M edicine , Emory U niversity, Atlanta, G A, USA. (Received 30 August 2004, revised 12 October 2004, accepted 25 October 2004) Eur. J. Biochem. 271, 4903–4908 (2004) Ó FEBS 2004 doi:10.1111/j.1432-1033.2004.04459.x heterozygous for the splicing abnormality [10]. In addition, the intensive analysis on novel mutations in human SSADH locus suggested that the missense mutations caused by point mutations, s mall insertionsand s malldeletions in t he genomic level would be causative of SSADH deficiency [11,12]. Despite the importance of SSADH in the metabolism of GABA, the structural studies of the enzyme have not yet been well in vestigated. We h ave purified and characterized an NAD + -dependent SSADH from bovine b rain [2], and found that the arginine residues are connected with catalytic activity of the enzyme [13]. Recently, we reported that a specific lysyl residue is located at or near the coenzyme binding site of the enzyme [14]. In the present study, we identified a regulatory site of the brain SSADH by a combination of l abeling with 4 -dimethylaminoazobenzene- 4-iodoacetamide (DABIA) and p eptide analysis. Materials and methods Materials NAD + , succinic semialdehyde, DABIA, EDTA, bovine serum albumin, trypsin (treated with tosylphenylalanylchlo- romethane), and 2-mercaptoethanol were purchased from Sigma (St Louis, MO, USA). CM-Sepharose, Blue-Seph- arose, and 5¢-AMP-Sepharose were obtained from Amer- sham Bioscience (Piscataway, NJ, USA). Bovine brains were obtained from Majang-dong Packing Company (Seoul, Korea). Purification of enzyme and enzymatic assays SSADH from bovine brain was purified according to a procedure previously described [2]. The procedure exploited four column chromatographic steps: C M-Sepharose, Blue- Sepharose, hydroxyapatite and 5¢-AMP-Sepharose. For precise measurement of enzymatic act ivity, the formation of NADH was measured by the increase in absorbance at 340 nm. All enzymatic assays were performed in duplicate and the initial velocity data was correlated with a s tandard assay mixture containing 30 l M succinic semialdehyde a nd 1m M NAD + in 0.1 M sodium pyrophosphate (pH 8 .4) a t 25 °C. One unit of enzyme was defined as the amount of enzyme required to reduce 1 lmol of NAD + per min at 25 °C. Protein concentrat ion was estimated by the Bradford procedure with a bovine s erum albumin standard [15]. Spectroscopic studies The purified enzyme (5 l M ) was incubated with 300 l M DABIA at 25 °C, in the dark, for 30 m in. At the end of incubation, the sample was dialyzed against 0.1 M potas- sium phosphate (pH 7 .0) a nd the absorption s pectra were recorded with a Kontron UVIKON 930 double beam spectrophotometer (Tegimenta, Rotkreuz, Switzerland) in the range 32 5–500 n m. Modification of succinic semialdehyde dehydrogenase with DABIA The purified e nzyme was dialyzed against 0.1 M potassium phosphate (pH 7.0), 1 m M EDTA, a nd then used immedi- ately. A total of 200 m M of DABIA was freshly prepared i n dimethylformamide and kept on ice. The final concentration of dimethylformamide in the incubation mixture was no more than 1% (v/v) and was found to have no effect on enzymatic activity. The i ncubation mixture (1 mL) con- tained S SADH (5 l M ), DABIA (100–400 l M )and0.1 M potassium phosphate (pH 7 .0). The reaction was initiated by addition of DABIA in the dark at 25 °C. At intervals after the initiation of inactivation, aliquots were withdrawn for the activity assay. Whenever possible a small sample volume (2 lL) was used t o minimize artifactual blank due to the t ransfer of DABIA. Protection experiments were performed in a similar manner except that the enzyme (5 l M ) was preincubated with a substrate SSA (3 m M ) or c oenzyme NAD + (3 m M ) at 25 °C for 30 min before the modification was initiated by the addition of DABIA. The amount of DABIA b ound to the enzyme was determined by measuring the increase in absorbance at 436 nm using a m olar extinction coefficient of 29 000 M )1 Æcm )1 [16]. Labeling and tryptic digestion of succinic semialdehyde dehydrogenase To identify the DABIA binding site, 2.9 mg of enzymes (50 l M ) w ere t reated with DABIA as d escribed previously [14,16]. Labeling of protein was conducted for 30 min in the dark at 25 °C. The excess reagent was removed by Sephadex G-25 superfine gel filtration. The solution was dried, dissolved by first adding 20 lLof50%(v/v)formicacid and then 600 lL water, transferred to a smaller tube suitable for enzymatic digestion a nd dried again. DABIA-labeled protein (20 nmol) was suspended in 0.5 m L of 0.1 M ammonium bicarbonate buffer (pH 8), and digested with trypsin, previously treated with tosylphenyl- alanylchloromethane, for 20 h at 37 °C. The substrate/ enzyme molar r atio was 50 : 1. Purification of DABIA-labeled cysteine-containing peptides To a 0.5 mL tryptic digest, 40 lL acetic acid was added and the precipitate was removed by centrifugation (10 000 g,20 min, 4 °C). Peptides i n the sample solution were lyophilized and separated by reverse-phase chromatography (LKB Instruments, Uppsala, Sweden ) using a Vydac C 18 column (0.46 · 25 cm). The separation was perform ed with a linear gradient from 0 to 70% B in 40 min at a flow rate of 0.8 m LÆmin )1 .EluantA:10m M potassium phosphate (pH 7 ) containing 2% dimethylformamide; eluant B: acetonitrile containing 4% dimethylformamide. Further purification was achieved b y rechromatograph- ing the peptides o n a Vydac C 18 column with a linear gradient of 0–60% B i n 60 min. Eluant A: 0.1% t rifluoro- acetic acid (pH 2.15); eluant B: 0.1% t rifluoroacetic a cid in acetonitrile/H 2 O (80 : 20, v/v). Cysteine derivatized with DABIA and the corresponding phenylthiohydantoin deriv- ative were prepared and characte rized according t o the procedure described by Chang et al. [17]. The latter was used as standard in the quantitative evaluation of other phenylthiohydantions obtained after automated Edman degradation. The absorption properties of the 4904 B. R. Lee et al.(Eur. J. Biochem. 271) Ó FEBS 2004 DABIA d erivative o f cysteine (molar extinction coefficient e ¼ 29 000 M )1 Æcm )1 at 436 nm) was used to determine the cysteine content of t he derivatized peptides. Amino acid analysis and peptide sequencing Peptides (6 nmol) were hydrolyzed for 24 h in 6 M HCl, containing 0.1% thioglycolic acid at 110 °C in vacuum. Amino acids derivatized with phenylisothiocyanate were identified and quantified by HPLC (Waters, Milford, MA, USA) Pico-Tag system, using a Nova-Pak C 18 column run at room temperature with a flow rate of 1 mLÆmin )1 . For the amino acid sequence analysis, the labeled peptide was subjected to automated Edman degradation on a Beckman Model 890M sequenator according to the manu- facturer’s instructions. Results Inactivation of succinic semialdehyde dehydrogenase by DABIA The r elevance of sulfhydryl groups in the catalytic activity of SSADH, was examined by reacting the enzyme with DABIA. DABIA, a chromophoric reagent, was chosen for our study for the following reasons: (a) it reacts with the S H groups of SSADH under native c onditions; (b) the labeled peptides and amino acid residues are easily monitored at 436 n m because of the large extinction coefficient of the chromophore; (c) i n a case to purify the peptides alkylated by DABIA, the derivatized peptides are more hydrophobic than unlabeled peptides, allowing their separation by reverse phase HPLC. Incubation of SSADH with increasing concentrations of DABIA r esulted i n a progressive d ecrease in its e nzymatic activity (Fig. 1 ). This inactivation followed pseudo first- order k inetics w ith c oncentrations of DABIA i n t he range 100–400 l M . The pseudo first-order rate constants, obtained at each DABIA concentration, were plotted as a function of DABIA concentration (Fig. 1, inset). The second-order rate constant for the inactivation of DABIA was 168 M )1 Æmin )1 , as determined from the slope of this plot. In an effort to demonstrate that DABIA is bound to sulfhydryl groups of SSADH, 5 l M of SSADH was incubated with or without 400 l M of DABIA at p H 7.0 in the dark for 30 min and absorption was monitored from 325 to 500 nm. The spectrum of DABIA-labeled enzyme showed a characteristic p eak at 4 36 nm (Fig. 2 , curve 2), which was absent from the spectrum o f the native enzyme (Fig. 2, curve 1). The absorption at 436 nm corresponds to a DABIA alkylated sulfhydryl g roup chromophore. The value for the incorporation of DABIA labeled on SSADH was measured using an extinction coefficient of 29 000 M )1 Æcm )1 at 436 n m. DABIA gave overall incorporation values of about 7.5 m ol per enzyme tetramer, indicating that 8 mols of sulfhydryl groups of SSADH were masked. The correlation between DABIA i ncorporation and SS ADH e nzyme activ- ity is shown in Fig. 3 . During the inactivation process, a linear relationship w as obse rved betw een DABIA a nd the loss of enzyme activity, which extrapolates to a stoichio- metry of 8.0 mol DABIA derivatives per mol enzyme tetramer, based on increased absorbance at 436 nm. The inactivation studies were carried out in the presence of substrate or coenzyme to define the site(s) modified by DABIA. The reaction between SSADH and DABIA was effectively prevented by incubating SSADH with the substrate, SSA, but not with coenzyme, NAD + (Table 1). Fig. 1. Determination o f the rate constant (K obs ) f or the inactivation o f SSADH at different concentrations of DABIA. The enzyme (5 l M )was incubated with 1 00 l M (d), 200 l M (s), 300 l M (j) a nd 400 l M (h) of DABIA in 0 .1 M potassium phosphate ( pH 7.0) at 25 °Cinthe dark. Aliquots withdrawn from the incubation mixtu res were tested for enzymatic activity. T he inset sh ows the d epen dence of the observed rate constant (K obs ) on DABIA concentratio n. Fig. 2. Absorption spectra of native (curve 1) and DABIA-treated (curve 2) SSADH. At the end of incubation, the absorption spectra were determined as desc ribed in M aterials and m ethod s. Ó FEBS 2004 Brain succinic semialdehyde dehydrogenase (Eur. J. Biochem. 271) 4905 These results suggest that the loss of SSADH enzymatic activity may be the result of the binding of DABIA to specific sulfhydryl groups located at or near the substrate binding site of SSADH. Isolation of modified peptides To identify the peptides modified by D ABIA, SSADH was treatedwithDABIAanddigestedwithtrypsinasdescribed above. After overnight trypsin digestion, the digested sample was loaded on to a reverse-phase column (Vydac C 18 ). Two peptides, designated I and II, were detected by monitoring the absorption spectrum at 436 nm (data not shown), indicating that the modification induced by DABIA was restricted to at least two amino acids in the SSADH subunits. Each peptide tagged with DABIA was further purified by a second chromatography through a Hypersil ODS column using a different solvent system as described previously [14,16]. A fter the second chromatography, two single pure peptides derivatized with DABIA were isolated from the originally labeled p eptides, respectively, a s shown in F ig. 4. Amino acid analysis and protein sequencing The amino acid analysis and s equence of p eptides I a nd II were examined and the observed sequences were found to be in reasonable agreement with the a mino acid compositions determined after the acid hydrolysis of each DABIA-labeled peptide (data not shown). The s toichiometric study (Fig. 3 ) and the analysis of amino acid sequence ( Table 2 ) showed that one cysteine residue in each peptide (peptides I and II) was labeled with DABIA. The amino acid sequence analyses of peptides I and II revealed that the peak fractions contained the Fig. 3. Stoichiometry of DABIA inactivation. SSADH (5 l M )was incubated with 400 l M of DABIA in 0.1 M potassium phosphate (pH 7.0) at 25 °C in the dark. The inactivation of SSADH is plotted as a f unction o f mol DABIA incorporated per mol e nzyme. Table 1. Inactivation of succinic semialdehyde dehydrogenase by DABIA. Enzyme (5 l M ), DABIA (300 l M ), NAD + (3 m M )andsuccinic semialdehyde (3 m M )wereused.Thedatarepresentthemeanofthree independent experiments with the difference expr essed as ± deviation. Reaction mixture Remaining activity (%) Enzyme 100 ± 3 Enzyme + DABIA 15 ± 2 Enzyme + NAD + + DABIA 23 ± 3 Enzyme + succinic semialdehyde + DABIA 91 ± 4 Fig. 4. Second chromatography of the tryptic peptides labeled with DABIA. Peptides I and II were purified by HPLC using a Vydac C 18 column and a linear gradient of ac etonitrile (0–60%) containing 5m M sodium phosphate ( pH 6.4) for 120 min at a flow rate of 0.5 mLÆmin )1 . Elution was monitored at 436 nm and the DABIA- labeled peptides ( I and II ) were s equenced by Edman degradation. Table 2. Sequences of the cysteine-containing tryptic peptides from succinic semialdehyde dehydrogenase. DABIA-Cys (Dys) was deter- mined as the DABIA phenylthiohydantoin (residues indicated in bold). Peptide Sequence I Val-Dys-Ser-Asn-Gln-Phe-Leu-Val-Gln-Arg II Glu-Val-Gly-Glu-Ala-Ile-Dys-Thr-Asp-Pro- Leu-Val-Ser-Lys 4906 B. R. Lee et al.(Eur. J. Biochem. 271) Ó FEBS 2004 amino acid s equences Val-Xaa-Ser-Asn-Gln-Phe-Leu-Val- Gln-Arg and Glu-Val-Gly-Glu-Ala-Ileu- Xaa-Thr-Asp-Pro- Leu-Val-Ser-Lys, respectively, where Xaa represents an assayable phenylthiohydantoin amino acid. This residue can be designated DABIA-Cys (Dys ),basedonaminoacid analysis. O f i nterest, the amino acid sequences of peptides I and II were found to be identical to regions of human SSADH, i.e. amino acids 341–350 and 266–279, respect- ively. Discussion Little is known about the chemistry of the active site of SSADH, partly because t he crystal structure of this enzyme is not available. Therefore, it is essential that a detailed structural desc ription of SSADH is elucidated. Previously, we purified the homotetramer SSADH from bovine brain homogenate [ 2]. Recently, an investigation o n the cat alytic role of specific amino a cid residues i n the enzyme i ndicated the involvement of a lysyl residue in its e nzymatic activity [14]; this conclusion was reached based on evidence obtained by chemically modifying SSADH with pyrid- oxal-5¢-phosphate, a specific lysine residue modifying rea- gent. In the present study, we identified at least one substrate binding domain in brain SSADH by combining DABIA labeling and peptide a nalysis. DABIA has been widely used in structural and functional studies to selectively label reactive cysteine residues in particular, which are ofte n directly involved in the catalytic mechanisms of active sites [16–18]. Haloacetamide deriva- tives such a s DABIA reac t with c ysteine via a S N 2reaction mechanism to give the corresponding carboxamidomethyl derivatives. The r eaction of h aloacetamide derivatives w ith cysteine is 20–100 times as rapid as with other cysteine- modifying reagents. In addition, the t wo benzene rings provide DABIA with a large extinction coefficient, which allows DABIA-labeled cysteine to be measured efficiently and rapidly [19]. The incubation of SSADH with increasing concentra- tions of DABIA resulted in a p rogressive reduction in enzyme activity (Fig. 1 ). The evidence for the specific modification o f cysteine r esidues by D ABIA was provided by monitoring the absorption of DABIA-alkylated cyste- ines at 436 nm ( Fig. 2). The nature of the i nhibitory effect exerted by DABIA was studied in detail. The possibility t hat DABIA inhibition is the result of the reaction of cysteine residues critically connected with catalysis was investigated by performing inhibition studies in the presence and absence of the substrate S SA or in the presence or absence of coenzyme NAD + . At pH 7.0, the inhibitory effect of DABIA was influenced by SSA a t 3.0 m M (Table 1) . The near complete protection afforded by SSA, strongly suggests that the inactivation occurred because of an interaction between DABIA and cysteine residues located at or near the substrate binding site of SSADH. In marked contrast to SSA, the coenzyme NAD + , did not afford any protection against DABIA i nactivation. Although d ifferences in the absorption spectra of native and DABIA-labeled SSADH were observed by absorption spectroscopy (Fig. 2), we have evide nce that the conform- ational changes of SSADH did not occur when it reacts with DABIA. We investigated these conformational changes indirectly by fluorometric anisotropy, but no differences in the anisotropies (A) of native (A ¼ 0.174) and modified enzyme (A ¼ 0.1 79) were observed. This observation demonstrates that the inactivation of SSADH occurs due to the interaction between DABIA and cysteine residues o n SSADH, and t hat it is not due to conformational changes of SSADH. During the inactivation process, a linear relationship was observed between DABIA and the l oss of enzyme a ctivity, which extrapolates to a stoichiometry of 8.0 m ol DABIA derivatives p er mol enzyme tetramer, based on increased absorbance at 436 n m (Fig. 3). There has been major controversy concerning SSADH protein structure. In the early 1970s, Cash et al. reported that SSADH was a dimeric protein of mass i dentical subunits [20], however, in the 1980s, Ryzlak & Pietruszko reported that SSADH was a tetrameric protein of mass nonidentical subunits [3], a finding never repeated in the aldehyde dehydrogenase literature. Our results support the cloning data of Chambliss et al . [8], that SSADH is a protein of ho motetrameric structure with mass identical subunits. Our previous puri- fication of SSADH from bovine brain showed that the SSADH is a tetramer c omposed of mass identical subunits, although there is minor variation in t he molecular mass of a single subun it [2]. This result is in keeping with t he notion that mammalian SSADH is a homotetramer, and also supports our conclusion that two cysteine residues per single subunit are l abeled by DABIA. To identify the site of inactivation, tryptic peptides containing DABIA-labeled cysteine were prepared. The results of sequence analysis (Table 2 ) showed that the modified residues correspond to the cysteine residues already identified in SSADH from human liver and rat brain [8]. Even though the amino acid composition and sequence of bovine brain SSADH have not been identified, the labeled cysteine residues in peptides I and I I correspon- ded to Cys342 and Cys272, respectively, of mammalian brain SSADH. Cys342 is conserved in all members o f the aldehyde dehydrogenase superfamily from bacteria to human, and is presumed to be located at an active site based on t he sequence homology with bovine aldehyde dehydrogenase, the three-dimensional structure of which has been solved [21]. This finding is consistent with our observation that the substrate, SSA, nearly completely blocked the DABIA-mediated in hibition of SSADH, but coenzyme NAD + did not. On the other hand, Cys272, found in peptide II, appears to be located near the coenzyme binding site. Sequence similarity between Cys272 and the aldehyde dehydrogen ase s uperfamily shows that Cys272 is located 12 amino acids away from Gly284, which is widely accepted to be a coenzyme binding site. In addition, coenzyme binding sites in bovine a ldehyde dehydrogenase have been shown to lie in an a-helical structure near the surface of the enzyme [21]. If the three-dimensional structure of SSADH is not much different from the structure of aldehyde dehydrogenase, the DABIA molecule bound to Cys272 seems to be located too far away from the coenzyme binding site to interfere the NAD + –protein interaction. In summary, the study presented here establishes that brain SSADH is inhibited by t he binding of DABIA to specific cysteine residues at or near the active site of the protein. Knowledge o f the interaction between DABIA and Ó FEBS 2004 Brain succinic semialdehyde dehydrogenase (Eur. J. Biochem. 271) 4907 SSADH may provide insights into approaches for the design of a new class of regulators, which do not resemble SSADH substrates. Acknowledgements This work was suppo rted by the 21st Century Brain Frontier Research Grant (M103KV010019-03K2201-019 10), and National Research Laboratory (NRL) Grant (M1-9911-00-0025 ) from the Ministry of Science and Technology, and in part by the Research Grant from Hallym University. References 1. Chambliss, K .L. & G ibson, K.M. (1992) Succinic s emialdehyde dehydrogenase from mamm alian brain: subunit analysis using polyclonal antiserum. Int. J. Biochem. 24, 1493–1499. 2. Lee, B.R., H on g, J.W., Yoo, B .K., Lee, S.J., Cho, S.W. & Choi, S.Y. (1995) Bovine brain succinic semialdehyde dehydrogenase: purification, kinetics and reactivity of lysyl residues connected with catalytic activity. Mol. Cells 5, 611–617. 3. Ryzlak, M.T. & Pietruszko, R. (1988) Human brain Ôhigh KmÕ aldehyde dehydrogenase: purification, characterization, and identification as N AD + -dependent succinic semialdehyde dehy- drogenase. Arch. B iochem. B iophys. 266, 386–396. 4. Jakobs, C., Jaeken, J. & Gibson, K.M. (1993) Inherited dis orders of GABA metabolism. J. Inherit. Metab. Dis. 16, 704–715. 5. Hogema, B.M., Akaboshi, S., Taylor, M., Solomons, G.S., Jakobs, C., S chutgens, R.B ., W ilcken, B., Worthington, S., Maropoulos, G., Grompe, M. & Gibson , K.M. ( 2001) Prenatal diagnosis o f su ccin ic semialdehyde dehy drogenase deficiency: Increased accuracy employing DNA, enzyme, and metabolic analyses. Mol. Genet. Metab. 72, 218–222. 6. Cho, S.W., Son g, M.S., Kim, Y.G., Kang, W.D., Choi, E .Y. & Choi, S.Y. (1993) Kinetics and m echanism of an NADPH- dependent succinic semialdehyde reductase from bovine brain. Eur. J. Biochem. 21 1 , 757–762. 7. Snead, O.C. (19 78) G amma hydroxybutyrate in the monkey. I. Electroencephalographic, behavioral, and pharmacokinetic stud- ies. N eurology 28 , 636–642. 8. Chambliss, K.L ., Caudle, D.L ., Hinson, D.D., Moomaw, C.R., Slaughter, C.A., Jakobs, C. & Gibson, K.M. (1995) Molecular cloning of the mature NAD(+)-dependent succinic semialde- hyde dehydrogenase from rat and human. cDNA isolation, evo- lutionary homology, and tissue expression. J. Biol. Chem. 270, 461–467. 9. Blasi,P.,Boyl,P.P.,Ledda,M.,Novelleto,A.,Gibson,K.M., Jakobs, C., Homega, B., Akaboshi,S.,Loreni,F.&Malaspina,P. (2002) Structure of h uman suc cinic se mialde hyde dehydroge nase gene: Identification of promoter region and alternatively processed isoforms. Mol. Genet. Met ab. 76, 348–362. 10. Chambliss, K.L., Hinson, D.D., Trettel, F., Malaspina, P., Novelletto, A., Jakobs, C. & Gibson, K.M. (1998) Two exon- skipping mutations as the molecular basis of succinic semialdehyde dehydrogenase deficiency (4-hydroxybutyric acid- uria). Am.J.Hum.Genet.63, 399–408. 11. Aoshima, T., K ajita, M., Sekido, Y., Ishiguro, Y., Tsuge, I., Kimura,M.,Yamaguchi,S.,Watanabe,K.,Shimokata,K.& Toshimitsu, N. (2002) Mutation analysis in a p atient with succinic semialdehyde deh ydrogen ase deficiency: a c ompound hetero- zygote with 103–121del and 146 0T>A of the ALDH5A1 ge ne. Human Heredity 53 , 42–44. 12. Akaboshi, S., Hogema, B.M., Novelletto, A., Malaspina, P., Salomons, G.S., Maropoulos, G.D., Jakob s, C., Grompe, M. & Gibson, K.M. (2003) Mutational spectrum of the s uccinic semi- aldehyde dehydrogenase (ALDH5A1) ge ne and functional ana- lysis o f 27 novel disease-causing mutations in patients with SSADH deficiency. Human M utatio n 22, 442–450. 13. Bahn,J.H.,Lee,B.R.,Jeon,S.G.,Jang,J.S.,Kim,C.K.,Jin,L.H., Park,J.,Cho,Y.J.,Cho,S.W.,Kwon,O.S.&Choi,S.Y.(2000) Brain succinic s emialdehyde dehydrogenase: r eaction of arginine residues connected w ith catalytic activities. J. Biochem. Mol. Biol. 33, 3 17–320. 14. Choi,S.Y.,Bahn,J.H.,Lee,B.Y.,Jeon,S.G.,Jang,J.S.,Kim, C.K.,Jin,L.H.,Kim,K.H.,Park,J.S.,Park,J.&Cho,S.W. (2001) Brain succinic semialdehyde dehydrogenase: identification of reactive lysyl residues labeled with pyridoxal-5¢-phosphate. J. Neurochem. 76, 919–925. 15. Bradford, M.M. (1976) A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-d ye binding. Anal. Biochem. 72, 248–254. 16. Kim, Y.T. & Churchich, J.E. (1989) S equence of t he c ysteinyl- containing pe ptides of 4-aminobutyrate aminotransferase. Iden- tification of sulfhydryl residues i n volved in intersubunit linkage. Eur. J. Biochem. 181, 397–401. 17. Chang, J.Y., Knecht, R. & Braun, D.G. (1983) A new method for the selective isolation of cysteine-containing peptides. Specific labeling of the thiol group with a hydrophobic chromophore. Biochem. J. 211, 163–171. 18. Cardamone, M., Aslunan, K., Brandon, M.R. & Puri, N.K. (1993) Identification of cys-containing peptides during peptide mapping of recombinant proteins. Pept. Res. 6, 2 42–248. 19. Lundblad, R.L. (1995) The modification of cysteine. In Techniques in Protein Modification pp. 63–83. C RC Press, Boca Raton, FL. 20. Cash, C .D., Ma itre, M . & Mandel, P. ( 1979) Purificati on f rom human brain and some properties of two NADPH-linked akde- hyde reductases which reduce succinic semialdehyde to 4- hydro- xybutyrate. J. Neurochem. 33 , 1169–1175. 21. Steinmetz, C.G., Xie, P., Weiner,H.&Hurley,T.D.(1997) Structure of m itochondrial aldehyde d ehydrogenase: the genetic component of ethanol a ve rsion. Structure 5, 701–711. 4908 B. R. Lee et al.(Eur. J. Biochem. 271) Ó FEBS 2004 . Brain succinic semialdehyde dehydrogenase Reactions of sulfhydryl residues connected with catalytic activity Byung Ryong Lee 1 ,. (1995) Bovine brain succinic semialdehyde dehydrogenase: purification, kinetics and reactivity of lysyl residues connected with catalytic activity. Mol.