dopamine inhibition of human tyrosine hydroxylase type 1 is controlled by the specific portion in...

Journal of Neurochemistry Lippincott Williams & Wilkins, Inc., Philadelphia 0 1999 International Society for Neurochemistry

Dopamine Inhibition of Human Tyrosine Hydroxylase Type 1 Is Controlled by the Specific Portion in the N-Terminus

of the Enzyme

Akira Nakashima, Keiji Mori, TTakahiro Suzuki, “Hideki Kurita, “Motohiko Otani, TToshiharu Nagatsu, and Akira Ota

Departments of Physiology and *Hygiene, School of Medicine, and +Division of Molecular Genetics, Institute f o r Comprehensive Medical Science, Fujita Health University, Aichi, Japan

Abstract: Tyrosine hydroxylase (TH), which converts L-tyrosine to L-DOPA, is a rate-limiting enzyme in the biosynthesis of catecholamines; its activity is regulated by feedback inhibition by catecholamine products includ- ing dopamine. To investigate the specific portion of the N-terminus of TH that determines the efficiency of dopamine inhibition, wild-type and N-terminal 3 5 , 38-, and 44-amino acid-deleted mutants (del-35, del-38, and del- 44, respectively) of human TH type l were expressed as a maltose binding protein fusion in Escherichia coli and purified as a tetrameric form by affinity and size-exclusion chromatography. The fused-form wild-type enzyme possessed almost the same specific enzymatic activity as the previously reported recombinant nonfused form. Al- though maximum velocities of all N-terminus-deleted forms were about one-fourth of the wild-type value, there was no difference in Michaelis constants for L-tyrosine or (6R)-(~-erythro-l’,2’-dihydroxypropyl)-2-amino-4-hydroxy- 5,6,7,8-tetrahydropteridine (GRBPH,) among the four enzymes. The iron contents incorporated into the three N-terminus-deleted mutants were significantly lower than that of wild type. However, there was no substantial difference in incorporated iron contents among the three mutants. The deletion of up to no less than 38 amino acid residues in the N-terminus made the enzyme more resis- tant to dopamine inhibition than the wild-type or del-35 TH form. Dopamine bound to the del-38 more than to the del-35 TH form. However, when incubation with dopamine was followed by further inhibition with the cofactor GRBPH, dopamine was expelled more readily from the del-38 than from the del-35 TH form. These observations suggest that the amino acid sequence G l ~ ~ ~ - A r g ~ ~ - A r g ~ ~ plays a key role in determining the competition between dopamine and GRBPH, and affects the efficiency of dopamine inhibition of the catalytic activity. Key Words: Tyrosine hydroxylase-N-Terminus-deleted mutant- Dopamine inhibition-Tetrahydrobiopterin-lron-Mal- tose binding protein. J. Neurochem. 72, 21 45-21 53 (1 999).

Tyrosine hydroxylase [TH; tyrosine 3-monooxygenase; L-tyrosine, tetrahydr0pterin:oxygen oxidoreductase

(3-hydroxylating); EC 1.14.16.21, which catalyzes the conversion of L-DOPA from L-tyrosine (Nagatsu et al., 1964), is a rate-limiting enzyme in the biosynthesis of catecholamines (Levitt et al., 1965). The enzyme requires a reduced pterin (Nagatsu et al., 1964), and (6R)-(~-erythro- 1 ,2’-dihydroxypropyl)-2-amino-4-hydroxy- 5,6,7,8-tebxhydropteridine (6R-tebxhydrobiopteri~ 6RBPH4) is the natural cofactor (Kaufman, 1963; Brenneman and Kaufman, 1964; Matsuura et al., 1985). TH also requires ferrous iron (Nagatsu et al., 1964; Shiman et al., 1971). TH consists of a catalytic domain (C-domain) and a regulatory domain (R-domain) (Abate and Joh, 1991). The C-domain is located at the C-terminal two-thirds of the molecule and binds the substrates (L-tyrosine and molecular oxygen) and the cofactor (6RBPH,). In contrast, the R-domain has been assigned to the N-terminal end (Hoeldtke and Kaufman, 1977; Abate et al., 1988; Abate and Joh, 1991) and has an important role in substrate specificity and in control of the catalytic activity (Haavik et al., 1990; Daubner et al., 1992; Lohse and Fitzpatrick, 1993; Martinez et al., 1996). Deletion studies have further defined the location of the regulatory domain to the N-terminus. Abate et al. (Abate et al., 1988; Abate and Joh, 1991) demonstrated by trypsin proteolysis experiments that the N-terminus of TH regulated cofactor binding. TH is inhibited by the end product catecholamines: Without preincubation, the inhibition requires high millimolar concentrations and is competi-

~~

Received September 14, 1998; revised manuscript received Decem- ber 28, 1998; accepted December 30, 1998.

Address correspondence and reprint requests to Dr. A. Ota at De- partment of Physiology, School of Medicine, Fujita Health University. Toyoake, Aichi 470-1192, Japan.

Abbreviations used: del-35, -38, and -44, N-terminal 35-, 38-, and 44-amino acid-deleted tyrosine hydroxylase mutants, respectively; hTH1, human tyrosine hydroxylase type 1; IPTG, isopropyl-0-D- thiogalactopyranoside; MBP, maltose binding protein; pBS, pBlue- script; 6RBPH,, (6R)-(~-erythro-1’,2’-dihydroxypropyl)-2-amino-4- hydroxy-5,6,7,8-tetrahydropteridine; SDS-PAGE, sodium dodecyl sulfate-polyacrylamide gel electrophoresis; TH, tyrosine hydroxylase.

2145

2146 A. NAKASHIMA ET AL.

tive with the pterin cofactor (Nagatsu et al., 1964, 1970; Udenfriend et al., 1965; Ikeda et al., 1966), but with preincubation, the inhibition requires low micromolar concentrations (Okuno and Fujisawa, 1991). The role of the catecholamines as feedback inhibitors is thought to be of physiological significance.

We recently showed that the N-terminal deletion of up to 39 amino acid residues was enough to abolish the inhibitory effect of M dopamine and suggested the possibility that the sequence G l ~ ~ ~ - A r g ~ ~ - A r g ~ ~ is the key sequence against the inhibitory effect of dopamine, using wild-type and N-terminus-deleted mutants of recombinant human TH expressed in Escherichia coli (Ota et al., 1997). However, we do not yet fully understand the mechanism that controls the dopamine inhibition in relation to the sequence and 6RBPH, or ferrous iron. Moreover, the experiment was performed with the supernatants of the cell lysates of E. coli, as it was impos- sible to purify the deleted mutants using conventional chromatography methods. To substantiate the mechanism, the purification of N-terminus-deleted mutants is inevitably needed. Therefore, to obtain pure N-terminus- deleted enzymes, we employed the pMAL-c2 expression vector to express wild-type and N-terminus-deleted mutants of human TH type 1 (hTHl), all of which were fused to maltose binding protein (MBP) in E. coli. Then, we purified the fusion proteins and analyzed their enzyme kinetics. Of four types of human TH, hTHl is an abundant species in the adrenal medulla and in the substantia nigra (Grima et al., 1987; Ichinose et al., 1994) and the most homologous to rat, mouse, and bovine TH. The purpose of this study was to elucidate how the N-terminal sequence G l ~ ~ ~ - A r g ~ ~ - A r g ~ ~ is involved in dopamine-induced inhibition of TH catalytic activity.

MATERIALS AND METHODS

Materials pBluescript (pBS) was purchased from Toyobo (Osaka, Ja-

pan). Ampli Tuq polymerase, restriction enzymes, T4 DNA ligase, and E. coli strain JM109 were from Takara (Kyoto, Japan). E. coli strain BL21(DE3) (F, omp T, r-, m-) was from Novagen (Madison, WI, U.S.A.). Expression vector pMAL-c2 and amylose resin were obtained from New England BioLabs (Beverly, MA, U.S.A.). 6RBPH, was synthesized as previously described (Oka et al., 1982). [7,8-3H]Dopamine was from Amersham Pharmacia Biotech (Buckinghamshire, U.K.). All other reagents used in this study were purchased from Sigma (St. Louis, MO, U.S.A.), Boehringer-Mannheim (Mannheim, Germany), Merck (Darmstadt, Germany), Wako (Osaka, Ja- pan), and Nacalai (Kyoto, Japan) and were of analytical grade.

Construction of wild-type and N-terminus-deleted hTHl cDNA

The full-length hTHl cDNA cloned into pBS (Kaneda et al., 1987) was used as a template for PCR to generate mutants with deleted N-terminal amino acid residues. The oligodeoxynucle- otide primers used are summarized in Table 1 . 5’ primers were designed to introduce an EcoRI site followed by the nucleotides encoding the amino acid sequence that is the cleavage site of enterokinase. Because the native form of the N-terminus of the

TABLE 1. Oligonucleotides used as primers in PCR

Wild-type and mutant forms

Wild type 5’ primer 4-21 5 ‘-GCGAATTCGATGATGA TGATAAACCCACCCCCGACGCC-

ACC-3’ del-35 form

5‘ primer 106-123 5 ‘ GCGAATTCGA TGA TGA TGA TAAA GGGCGCAGGCAGAG-

CCTC-3’ del-38 form

5’ primer 115-132 5 ’ GCGAATTCGA TGA TGA TGA TAAACAGAGCCTCATCGAG-

GAC-3‘ del-44 form

5’ orimer 133-150 5‘-GCGAATTCGA TGA TGA TGA TAAA GCCCGCAAGGAGCG-

GGAG-3’ 3’ primer 350-335 5 ‘-GCrnTCCGGCCGGGTCTCTAGAT-3’

~

The nucleotide residues of the primers are numbered in the 5’ to 3’ direction starting from the first residue of the ATG triplet, which codes for the initiation Met of full-length hTHl cDNA. EcoRI and BumHI restriction sites are underlined in the 5’ and 3’ primers, respectively. Nucleotides written in bold italic letters represent the sequence coding for the amino acid sequence that is the digestive site of enterokinase. For terminology of the mutants, see text.

hTHl molecule is cleaved at an N-terminal methionine (Fuji- sawa and Okuno, 1987), the cDNA lacking the first ATG triplet was constructed and referred to as the wild type. The mutants missing the first N-tenninal35, 38, and 44 amino acid residues are referred to as del-35, del-38, and del-44 forms, respectively. The PCR procedure for wild-type, del-35, del-38, and del-44 forms involved 25 cycles of denaturation at 95°C for 1 min, annealing at 55°C for 1 min, and polymerization at 72°C for 1 min using a model PJ2000 thermocycler (Perkin-Elmer Cetus, Norwalk, CT, U.S.A.). The amplified DNAs were digested with EcoRI and BamHI, and the resulting EcoRI-BamHI fragments were inserted by ligation into the pMAL-c2 vector. pMAL-c2, an E. coli cloning vector used in protein fusion and purification systems, is designed to create fusion between the cloned gene and the E. coli ma1 E gene. The pMAL-c2 containing the EcoRI-BamHI fragment of PCR products was used to trans- form E. coli strain JM109 according to the standard method (Sambrook et al., 1989). After screening the transformants, we purified positive mutant clones and confirmed their DNA se- quences by using a model 373A DNA Sequencing System (Applied Biosystems, Foster City, CA, U.S.A.). For construction of cDNA for wild-type, del-35, and del-38 forms, pMAL-c2 containing the EcoRI-BamHI fragment of PCR product was linearized by PstI digestion; the PstI-PstI fragment derived from the aforementioned pBS containing full- length hTHl cDNA was inserted into the pMAL-c2 by ligation to introduce central and C-terminal regions. The method to construct cDNA for the del-44 form was essentially the same as for wild-type, del-35, and del-38 forms except for the linear- ization by XbaI and the usage of the XbaI-XbaI fragment for the insertion. These cDNA constructs were employed to trans- form E. coli strain BL21(DE3).

J. Neurochem., Vol. 72, No. 5, 1999

N-TERMINUS OF TH REGULATES DOPAMINE INHIBITION 2147

Expression and purification of wild-type and N-terminus-deleted hTHl

The expression of the fused hTHl molecule in BL21(DE3) was performed as previously described (Nasrin et al., 1994) with minor changes. In brief, BL21(DE3) containing wild-type or the mutant's expression vector was grown in Luria-Bertani medium containing 50 pg/ml ampicillin and 0.1 mM ferrous ammonium sulfate at 37°C. Isopropyl-P-D-thiogalactopyranoside (IPTG) was added at A550 = 0.8 to a final concentration of 0.1 mM, and incubation continued for 4 h. After incubation, the cells were collected by centrifugation, washed twice with 50 mM Tris-HC1 buffer (pH 7.4), and resuspended in 25 mM Tris-HC1 buffer (pH 7.3) containing 8% sucrose and 1 mM dithiothreitol. The cells were disrupted by sonication on ice by six cycles of 30-s bursts at 2-min intervals at 50 W with an Ultrasonics power sonifier (Branson, Plainview, NY, U.S.A.). The crude cell lysates were centrifuged at 12,000 g for 10 rnin at 4"C, and the supernatants were stocked at -80°C until used.

Next, the fusion proteins expressed in BL21(DE3) were isolated by affinity chromatography as previously described (Guan et al., 1988). All the procedures described below for the purification were performed at 4°C. The lysate supernatants were diluted 1:10 with column buffer (10 mM Tris-HC1, pH 7.4, containing 0.2 M NaCI, 1 mM EDTA, and 0.2 mM phe- nylmethylsulfonyl fluoride) and applied to an amylose resin column (2 cm i.d. X 4 cm). The column was washed with 10 bed volumes of column buffer, and then the fusion protein was eluted with column buffer containing 10 mM maltose. The eluate was dialyzed against 10 mM Tris-HC1 buffer (pH 7.4) containing 0.2 M NaCl and 1 mM EDTA. Then, the dialysates were run through a Superose 6HR 10/30 column (Amersham Pharmacia Biotech, Uppsala, Sweden) equilibrated with 10 mM Tris-HC1 buffer (pH 7.4) containing 0.2 M NaCl and 1 mM EDTA at a flow rate of 0.5 ml/min and fractionated. The absorbance was monitored at 280 nm. The values for molecular mass of the proteins were estimated based on the elution positions of the standard molecular mass markers supplied in a high molecular weight gel filtration calibration kit (Amersham Pharmacia Biotech). The eluate through the amylose resin and the fraction obtained by size-exclusion chromatography were analyzed by 12.5% sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). The proteins that migrated into the gel were visualized by staining with Coomassie Brilliant Blue R-250.

Assay of catalytic activity of enzymes Catalytic activity of TH was assayed by HPLC measure-

ments of the product L-DOPA (Nagatsu et al., 1979). With the purified fusion protein (4-18 pg/ml), the reaction was initiated by the addition of 50 p1 of protein solution to the reaction mixture (150 pl), which contained 67 mM HEPES (pH 7.7), 133 mM P-mercaptoethanol, 0.27 mg/ml catalase, L-tyrosine, and 6RBPH4. Samples were incubated at 37°C for 10 min under air for the data to be given in the initial velocity. Under these conditions, TH activity was linear for at least 15 min. The resulting L-DOPA was isolated by use of an HPLC apparatus (Shimadzu, Kyoto, Japan) equipped with a Nucleosil 7C,, column (4.6 i.d. X 250 mm; GL Sciences, Tokyo, Japan) equilibrated with 0.1 M sodium phosphate buffer (pH 3.5) containing 1% methanol and 4 fl EDTA and measured with an electrochemical detector (ECD-100; EiCOM, Kyoto, Japan).

In the case of preincubation with dopamine, the enzymes (35 pg/ml) were preincubated with various concentrations of dopamine in 25 p l of 50 mM Tris-HCI buffer (pH 7.4) containing 0.2 M NaCl and 1 mM EDTA at 30°C for 20 min. The assay

was started by the addition of 175 p1 of a mixture containing 57 mM HEPES (pH 7.7), 114 mM P-mercaptoethanol, 0.23 mg/ml catalase, 0.057 mM L-tyrosine, and 0.23 mM 6RBPH,, and then samples were incubated at 37°C for 10 min under air. The final concentrations of L-tyrosine and 6RBPH4 were 0.05 and 0.2 mM, respectively.

Measurement of iron contents in enzymes Iron contents incorporated into the purified fused-form en-

zymes were determined by atomic absorption using a polarized Zeeman atomic absorption spectrophotometer type 2-8 100 (Hi- tachi, Tokyo, Japan).

Measurement of dopamine binding to del-35 and del-38 forms

The binding of dopamine to purified del-35 and del-38 fused forms was assayed as previously described (Ribeiro et al., 1992) with minor modifications. The binding of radiolabeled dopamine in a total volume of 50 p1 at 30°C for 20 min was initiated by the addition of 100 pmol of the enzyme (subunit) to 440,000 cpm of [7,8-3H]dopamine with enough nonradiolabeled dopamine to produce the desired concentrations in 50 mM Tris-HC1 buffer (pH 7.4) containing 0.2 M NaCl and 1 mM EDTA; this binding mixture was applied to a Micro Bio-Spin 6 column (Bio-Rad) that had been equilibrated with 10 mM Tris-HC1 buffer (pH 7.4) in advance. The bound dopamine was separated from free dopamine by centrifugation of this small gel filtration column, and its radioactivity was measured using a liquid scintillation counter type LS 1801 (Beckman Instru- ments, Fullerton, CA, U.S.A.). For investigating dopamine binding at the end of enzymatic catalysis, preincubation of enzymes with radiolabeled dopamine in 25 pl of 50 mM Tris-HC1 buffer (pH 7.4) containing 0.2 M NaCl and 1 mM EDTA at 30°C for 20 min was followed by the addition of a 45-pl reaction mixture for the assay of TH activity (130 mM HEPES, pH 7.7,260 mM P-mercaptoethanol, 0.53 mg/ml catalase, 0.13 mM L-tyrosine, and 0.53 mM 6RBPH4) and incubated further at 37°C for 10 rnin in the air. Although the total volume of this mixture was 70 p1, the final concentrations of each ingredient in the assay solution were the same as those used for the actual measurement of TH activity. The dopamine bound to the enzyme at the end of incubation was separated from free dopamine, and its radioactivity was measured as mentioned above. Next, the effects of the components of the assay mixture (6RBPH4 and L-tyrosine) and the reaction product L-DOPA on dopamine binding to the enzyme were examined. Subsequent to the preincubation of enzyme with the mixture of radiolabeled dopamine and 2.5 p M nonradiolabeled dopamine in 40 pl of 50 mM Tris-HC1 buffer (pH 7.4) containing 0.2 M NaCl and 1 mM EDTA at 30°C for 20 min, 10 pl of 6RBPH4, L-tyrosine, or L-DOPA in varying concentrations was added to the preincubation mixture and incubated further at 37°C for 10 min in the air. The final concentrations of these three substances used in the assay were 100, 200, and 1,000 pM. The radioactivity of the bound dopamine at the end of incubation was measured as mentioned above.

Protein measurement Protein concentration was measured by the dye-binding

method using the Bio-Rad Protein Assay Kit (Bradford, 1976) with bovine serum albumin as a standard.

Statistics Data were expressed as the means 2 SEM of three to five

determinations for each group. Student's unpaired t test was



(A) sDs-pAGE wild-type del-35 del-38 del-44 ---- kDa A B C A B C A B C A B C 94- 67, 43,

30- 20*

14*

(B) Size-exclusion chromatography

10 15 Volume (ml)

FIG. 1. A SDS-PAGE of the proteins through the stages of purification. Lanes A, the lysate supernatant of B E 1 (DE3); lanes B, eluate from the amylose resin; lanes C, the 520-kDa fraction isolated by size-exclusion chromatography on a Superose 6HR 10/30 (the peak indicated by the solid bar in B). B: Size-exclusion chromatogram of MBP-hTH1 fusion protein.

used to compare mean differences. A level of p < 0.05 was accepted as statistically significant.

RESULTS

Purification of recombinant hTHl expressed in E. coli The supernatants of the crude cell lysates of the

BL21(DE3) induced by IPTG possessed the TH specific activity for the conversion of L-tyrosine to L-DOPA in the presence of 0.2 mM L-tyrosine and 1 mM 6RBPH4. The generation of L-DOPA by wild-type, del-35, del-38, and del-44 forms was 75,46,39, and 3 1 nmol/min/mg of protein, respectively. In addition, a major band of - 110 kDa was observed on SDS-PAGE of the lysate supernatants of the wild-type and three mutants (Fig. lA, lanes A). It can be concluded that the fusion proteins were obtained in high yields in soluble forms with high TH specific activities even in the cell lysates.

The supernatants of the lysates of BL21(DE3) were applied to an amylose resin column, and then the ad- sorbed molecules were eluted by 10 mM maltose. The specific activities of the eluates of wild-type, del-35, del-38, and del-44 forms were 121, 72, 75, and 77 nmol/min/mg of protein, respectively, in the presence of 0.2 mM L-tyrosine and 1 mM 6RBPH4. The eluates gave a major band with a molecular mass of -110 kDa on SDS-PAGE; the purities of the eluates of wild-type, del-35, del-38, and del-44 forms, determined by densi- tograms, were 85, 76, 77, and 68% (Fig. lA, lanes B),

respectively. The recovery was -12-15% of the total lysate protein.

To collect the tetrameric form of the fusion protein, we applied the eluates from the amylose resin column to a Superose 6HR 10/30 column. As shown in Fig. 1, the size-exclusion chromatograms of the eluates exhibited a major peak of 520 kDa and several minor peaks; i.e., void volume, a shoulder-like 260-kDa peak (observed only for the del-44 form), and low molecular mass peaks. The resulting 520-kDa peaks (indicated by the bar in Fig. 1B) were collected; their TH specific activities, measured in the presence of 0.2 mM L-tyrosine and 1 mM 6RBPH4, were 947, 164, 224, and 225 nmol/min/mg of protein for wild-type, del-35, del-38, and del-44 forms, in this order. These values were remarkably higher than those from other minor fractions. Moreover, the major 520-kDa peak gave rise to a band with a molecular mass of - 110 kDa on SDS-PAGE, and the purity of the band was -90% (Fig. lA, lanes C ) . The recovery was 4-8% of the whole protein contained in the supernatant of the crude cell lysate and the purification -10-fold. Because the amino acid sequence (asparagine-rich region) to connect MBP with the hTHl molecule exhibits a linear structure, the fusion protein molecule might be expected to exhibit a higher molecular mass on size-exclusion chromatography. Therefore, we concluded that the fusion protein in the 520-kDa fraction existed in the tetrameric form. We used the 520-kDa fraction as an enzyme sample in the following experiments, if not stated.

Catalytic activities of purified enzymes Kinetic properties of wild type and mutants. Kinetic

parameters of wild-type, del-35, del-38, and del-44 forms were measured with L-tyrosine as a substrate and 6RBPH4 as a cofactor. When the dependency of the catalytic activity on the concentration of the substrate was examined using various doses of L-tyrosine from 0.01 to 0.20 mM with 1 mM 6RBPH,, substrate inhibition was observed above 0.05-0.10 mM L-tyrosine for all enzyme samples when the data were plotted according to Michaelis-Menten (Fig. 2). Michaelis constant (K,) and maximum velocity (V,,) were determined by Line- weaver-Burk plots using the points below 0.05 mM L-tyrosine on Fig. 2 where there was no substrate inhibition (Fig. 3). Calculated kinetic parameters are shown in Table 2. The K , values for L-tyrosine ranged from 22 to 32 pA4 and did not differ among the enzymes. On the contrary, the V,,, values for the N-terminus-deleted mutants were about one-fourth of that for wild-type form (328-431 nmoYmin/mg of protein for mutants vs. 1,400 nmol/min/mg of protein for wild type; p < 0.05).

Next, the dependency of the catalytic activity on the concentration of 6RBPH4 was examined by varying the concentration of 6RBPH4 from 0.03 to 1.0 mM with 0.05 mM L-tyrosine. Lineweaver-Burk plots as a function of the concentration of 6RBPH4 gave a straight line for each enzyme (Fig. 4). In this case, the respective K , values for 6RBPH, among all enzymes were indistinguishable within experimental error. On the other hand, all the V,,,

.I. Neurochem., Vol. 72, No. 5, 1999


1500- h .-

-0- wild-type -c- del-35 t del-38

I I I I 0 0.05 0.10 0.15 0.20

L-tyrosinc (mM)

FIG. 2. Activity of wild-type (filled circles), del-35 (open circles), del-38 (filled triangles), and del-44 (open triangles) forms of TH as a function of L-tyrosine concentration. The catalytic activities were measured in duplicate using the assay mixture containing various concentrations of L-tyrosine. The cofactor GRBPH, was kept constant at 1 mM. The data are representative of three independent experiments.

values of the mutants were about one-fourth of the V,,, value of the wild type. These kinetic parameters for 6RBPH4 are also summarized in Table 2.

Inhibition of catalytic activities of wild type and mutants by dopamine. The enzymes were incubated with various concentrations of dopamine at 30°C for 20 min, and the catalytic activities of the mixtures were measured in the presence of 0.2 mM 6RBPH4 and 0.05 mM L-tyrosine (Fig. 5). The preincubation with dopamine reduced the catalytic activities in a dose-dependent way.

0.005

-&dkZ?L -50 0 50 100 150 200

1/ [ L-tyrosinel (mM) -' FIG. 3. Lineweaver-Burk plots of the activity of wild-type (filled circles), del-35 (open circles), del-38 (filled triangles), and del-44 (open triangles) forms of TH as a function of L-tyrosine concentration. The catalytic activities were measured by use of assay mixtures containing various concentrations of L-tyrosine. The cofactor GRBPH, was kept constant at 1 mM. Lineweaver-Burk plots were obtained using the points below 0.05 mM L-tyrosine, where there was no substrate inhibition on Fig. 2. 1/V is the reciprocal of the rate measured (nmol/min/mg of protein); 1/ [~- tyrosine] is the reciprocal of the concentration of the substrate L-tyrosine (mM). Each point on the Lineweaver-Burk plots was determined in duplicate. The Lineweaver-Burk plots shown are representative of those obtained from three independent experiments.

TABLE 2. Steady-state kinetic parameters of wild-type and three mutant enzymes as function of concentration of

L-tyrosine or 6RBPH4

L-Tyrosine 6RBPH,

Vmax Vmax K, (nmol/min/mg K, (nmol/min/mg (@I) of protein) (CLM) of protein)

Wild type 25 -C 2 1,400 ? 272 70 2 9 870 2 185 del-35 32 2 6 372 2 95" 52 ? 4 218? 13" del-38 26 2 1 431 -C 83" 61 ? 3 368 2 39 del-44 22 t 3 328 5 61" 73 2 9 218% 6"

K,,, and V,,, values for L-tyrosine were determined using the points below 0.05 mM L-tyrosine, where there was no substrate inhibition. The concentration of 6RBPH, was set constant at 1 mM for the measurement of catalytic activity. K , and V,,, values for 6RBPH, were determined at 0.05 mM L-tyrosine. The values represent the means 2 SEM from three independent experiments, each of which was performed in duplicate.

" p < 0.05, significantly lower than that of wild type.

Preincubation with lop4 M dopamine gave -95% loss of the activity of wild-type and del-35 forms, whereas -80% of the catalytic activity was lost for the del-38 and del-44 forms. At the concentrations of and M, dopamine inhibited the catalytic activity of wild-type and del-35 by -30% more than that of del-38 and del-44. As a whole, dopamine inhibited wild-type and del-35 more effectively than the del-38 and del-44.

Iron contents of wild type and mutants Iron contents incorporated into the enzymes are sum-

marized in Table 3. The iron contents incorporated into

-A- deb38 - del-44

-20 -10 0 10 20 30 40

1 I I BRBPH,J (mM)-'

FIG. 4. Lineweaver-Burk plots of the activity of wild-type (filled circles), del-35 (open circles), del-38 (filled triangles), and del-44 (open triangles) forms of TH as a function of GRBPH, concentration. The catalytic activities were measured in the assay mixture containing various concentrations of GRBPH,. The substrate L-tyrosine was kept constant at 0.05 mM. 1/V is the reciprocal of the rate measured (nmol/min/mg of protein); l/[GRBPH,] is the reciprocal of the concentration of the cofactor GRBPH, (mM). Each point on the Lineweaver-Burk plots was determined in duplicate. The Lineweaver-Burk plots shown are representative of those obtained from three independent experiments.



-A- del-38

.- .; so 2

2s

n o 10-7 10-6 10-5 10-4

Dopamine (M)

FIG. 5. Effect of preincubation with dopamine on the catalytic activities of wild-type (filled circles), del-35 (open circles), del-38 (filled triangles), and del-44 (open triangles) forms of TH. The catalytic activities were measured in duplicate as described in Materials and Methods. The experiments were carried out three times independently, and respective data are displayed as mean 2 SEM (bars) values. All values marked with asterisks were significantly different from that of wild type: *p < 0.05, **p < 0.01, ***p < 0.005, ****p < 0.001.

the three N-terminus-deleted mutants were significantly lower than that incorporated into the wild type (0.74- 0.86 mol/mol for mutants vs. 1.24 mol/mol for the wild type; p < 0.01 or p < 0.05). However, there were no significant differences in the incorporated iron contents among three N-terminus-deleted mutants.

Dopamine binding to del-35 and del-38 The del-35 and del-38 forms were mixed with [7,8-

3H]dopamine containing various concentrations of nonradiolabeled dopamine at 30°C for 20 min, and the amounts of dopamine bound to these mutants were quan- titated. The saturation binding curves and corresponding Scatchard plots for the del-35 and del-38 forms are shown in Fig. 6A. This analysis revealed the following results: (a) The maximum binding (Bmax) of dopamine with del-35 was smaller than that with del-38 (del-35: 0.108 5 0.008 mol/mol subunit vs. del-38: 0.191 ? 0.016 mol/mol subunit; p < 0.05); (b) the dissociation constant (K,,) of del-35 was larger than that of del-38 (del-35: 2.86 t 0.25 p M vs. del-38: 1.14 5~ 0.17 pM; p < 0.01). However, these differences between the del-35 and del-38 forms vanished at the end of the following

TABLE 3. Iron contents of wild-type and three mutant enzymes

Fehbuni t (mollmol)

Wild type del-35 del-38 del-44

1.24 L 0.08 0.81 ? 0.026 0.86 2 0.08" 0.74 ? 0.10"

~

Iron contents are expressed as Felsubunit (moUmo1). The values represent the means ? SEM from three independent experiments, each of which was performed in triplicate.

" p < 0.05, h p < 0.01, significantly lower than that of wild type.

E - i 0.10

C

E 5 0.05 J

= c = o V

0 2 4 6 8 Dopamine (pM)

(B) z 0.05 - del-35

-4- del-38 2 0.04 A

s o J 9 , , , , 0 2 4 6 8

Dopamine (pill)

FIG. 6. Saturation curve for the binding of [7,8-3H]dopamine to del-35 (open circles) and del-38 (filled triangles) forms of TH. A Saturation binding curves of dopamine to del-35 or to del-38 TH forms and the resulting Scatchard plots are shown. [7,8-3H]Do- pamine was incubated with the del-35 or del-38 forms, and then bound dopamine was separated from free dopamine. B, bound dopamine; B/F, ratio of bound and free dopamine. The data shown are representative of three separate experiments. B: The incubation of [7,8-3H]dopamine with del-35 or del-38 TH forms was followed by further incubation with the assay mixture for 10 min. Then bound dopamine was separated from free dopamine. The procedures are described in detail in the text. The data shown are representative of three separate experiments performed in duplicate.

simulation of enzymatic reaction, as indicated by the saturation binding curve in Fig. 6B. The amounts of dopamine that bound to the del-35 and del-38 forms at the end of the incubation with 6RBPH4 decreased in a dose-dependent way, A concentration of 100 p M 6RBPH, was enough to reduce the amounts of dopamine bound to these enzymes (Fig. 7). The ratio of the amount of bound dopamine to that in the absence of 6RBPH4 was compared between the del-35 and del-38 forms. The ratio obtained for del-38 was significantly lower than that obtained for del-35 when incubated with 6RBPH4 (Fig. 7). L-DOPA only at the highest concentration examined (1,000 pM) reduced the amounts of dopamine bound to these enzymes. In contrast to 6RBPH,, L-DOPA did not give rise to any difference between del-35 and del-38 in dopamine binding (data not shown). L-Tyrosine did not reduce the amounts of dopamine bound to del-35 and del-38 at the concentrations examined (data not shown).



0 250 500 750 1000

6RBPH, (FM)

FIG. 7. Effect of GRBPH, on the binding of dopamine to del-35 (open circles) and del-38 (filled triangles) forms of TH. The data are plotted as a function of the final concentration of GRBPH,. The data are expressed as mean 2 SEM (bars) values of three separate experiments performed in duplicate. The values were compared between del-35 and del-38 TH forms. All values of the del-38 form marked with asterisks were significantly lower than those of the del-35 form obtained at the same concentration of GRBPH,: *p < 0.05, ***p < 0.005.

DISCUSSION

There have already been several reports on the purification of recombinant TH expressed in E. coli (Le Bourdellbs et al., 1991; Nasrin et al., 1994). In our previous work, we also expressed the N-terminus-deleted mutants of hTHl in E. coli by using the pET3c vector and estimated that the sequence G l ~ ~ ~ - A r g ~ ~ - A r g ~ ~ of the N-terminus might affect the dopamine-induced enzyme inhibition (Ota et al., 1996, 1997). Because the purification of N-terminus-deleted mutants was inevitably needed to define the role of the sequence, we tried to purify the N-terminus-deleted mutants of hTHl by size- exclusion chromatography and affinity chromatography on a heparin-Sepharose column as previously described (Nasrin et al., 1994). However, the N-terminus-deleted mutant molecules aggregated, probably due to improper folding, and they were eluted in the “void volume” by size-exclusion chromatography.

On the other hand, many reports have accumulated that indicate the pMAL-c2 vector to be highly suitable to stably express a recombinant protein as a fusion protein with MBP in E. coli and such fusion proteins to be easily purified with high yield and high purity (Gum et al., 1988; Martinez et al., 1995; Doskeland et al., 1996). Especially, Martinez et al. (1995) expressed the recombinant human phenylalanine hydroxylase using the pMAL expression system and succeeded in purifying the enzyme as a tetrameric form. Therefore, we adopted the pMAL-c2 vector to express wild-type and N-terminus- deleted mutants of hTHl and to purify the fusion proteins. The preparations of the recombinant hTHl wild- type, del-35, del-38, and del-44 forms as fusion proteins with MBP revealed a high degree of homogeneity and had a subunit molecular mass of 110 kDa and tetrameric form made up of four subunits (Fig. 1). The kinetic parameters, namely, K , and V,,, of fused wild-type enzyme expressed and purified in this experiment were

similar to those of the native types of rat TH (Wang et al., 1991) and human TH (Le Bourdellks et al., 1991), both of which were produced by recombinant techniques and purified as a tetrameric form (Table 2); in contrast, lower K, and V,,, values for 6RBPH4 were recently reported for recombinant hTHl (Alterio et al., 1998).

The L-tyrosine saturation curve of the fused wild-type enzyme was found to show strong substrate inhibition above 50-100 p M L-tyrosine, which means that the fused enzymes also behaved in the same way as reported for TH isolated from the adrenal gland (Shiman et al., 1971) or brain tissue (Katz et al., 1976) or for TH expressed in recombinant systems (Wang et al., 1991). Thus, the pMAL-c2 fusion protein expression system was successful in producing catalytically active hTH 1 with a tetrameric form. In the following experiments, the wild-type and mutant enzymes were analyzed without cleavage by enterokinase, which allowed faster and less complicated purification of the mutant proteins.

There are several pieces of evidence about the affinity of L-tyrosine and 6RBPH4 for TH. The removal of the N-terminal 154 amino acids of rat TH by proteolysis was reported to cause a change in the K, value for L-tyrosine from 40 to 16 p M (Abate and Joh, 1991). In addition, the K, value for phenylalanine and the K, value for L-tyrosine of the N-terminus 155-amino acid-deleted form at rat TH were indistinguishable (Daubner et al., 1997). As shown in Table 2, the removal of the N- terminus of hTHl up to 44 amino acids did not cause any change in the K , values for L-tyrosine. Therefore, it is suggested that the specific portion to determine the affinity of the enzyme for the substrate L-tyrosine exists in the N-terminal45-154 amino acids of the TH molecule. The K, values for 6RBPH4 as well as L-tyrosine were not affected by the removal of the N-terminus up to 44 amino acids. Pro327 in rat TH was shown to be a key amino acid residue that can modify the binding of 6RBPH4. Substitution of Leu for Pro327 in rat TH produced a molecule with a K , for 6RBPH4 20-fold higher than that of the wild-type molecule (Quinsey et al., 1996). This report as well as ours imply that the deter- minants of the binding affinity of the 6RBPH4 exist mainly in the C-domain.

In this study, the dopamine-induced inhibition of the catalytic activity occurred with the wild-type and del-35 forms to a higher degree than with del-38 and del-44 (Fig. 5), which suggests that the del-35 and del-38 forms behaved distinguishably in the competition between 6RBPH4 and dopamine. On the other hand, there was no substantial difference in the contents of incorporated iron between the del-35 and del-38 forms (Table 3). These results suggest that there should be no difference in the geometry surrounding the iron, which is supposed to localize in the active site of the catalytic domain (Good- will et al., 1997). In addition, there was no difference in the K, values for L-tyrosine and 6RBPH4 between del-35 and del-38 (Table 2). Collectively, the difference in the dopamine-induced inhibition of the catalytic activity



should not be attributed to the structure of the catalytic domain of these two mutant enzymes.

Therefore, we hypothesize that the positive charges of the Arg3'-Arg3' sequence contribute to the accessibility of dopamine to or to the expulsion of dopamine from the iron positioned in the active site, as the N-terminus is expected to be localized near the active site (Goodwill et al., 1997). We did not expect that dopamine would bind more to del-38 than to del-35 in the absence of a com- petitor (as the case of preincubation of the enzyme with dopamine) (Fig. 6A). However, at the end of the simulation of enzymatic catalysis done by mixing dopamine- bound enzyme with the assay mixture, dopamine binding with the del-35 and del-38 forms attained an equal level (Fig. 6B). Moreover, the expulsion of dopamine from the enzyme molecule occurred with the del-38 form more readily than with del-35 when mixed with 6RBPH4 alone (Fig. 7). In other words, these observations indicate that 6RBPH4 was more potent in expelling dopamine from del-38 than from del-35 and coincide well with the observation that the catalytic activity of del-38 was less inhibited by dopamine than was that of del-35. It should be noted that dopamine binding was not affected at all when the preincubation of N-terminus-deleted TH with dopamine was followed by the addition of L-tyrosine. Collectively, our working hypothesis that the amino acid sequence G l ~ ~ ~ - A r g ~ ~ - A r g ~ ' has the key role in the competition of dopamine and 6RBPH, on the TH molecule and finally determines the efficiency of dopamine- induced inhibition of the catalytic activities was amply substantiated by this dopamine binding experiment. Re- cent physicochemical studies focusing on the redox states of iron incorporated into TH molecule have clar- ified precisely the mechanisms by which dopamine che- lates iron at the active site of the enzyme. In addition, the trapping of the active site iron in the ferric state, the binding mode of 6RBPH4 with iron, and the reduction of ferric iron by 6RBPH4 during catalytic turnover have been extensively characterized (Michaud-Soret et al., 1995; Martinez et al., 1996; Meyer-Klaucke et al., 1996; Ramsey et al., 1996). More recently, the differential binding of dopamine to the ferric and ferrous state TH was clearly demonstrated (Ramsey and Fitzpatrick, 1998). As addressed by such a series of investigations, studies on the regulation of the redox states of iron incorporated into the N-terminus-deleted hTH 1 should be of special interest.

In conclusion, we present data indicating that the sequence G l ~ ~ ~ - A r g ~ ~ - A r g ~ ' affects the competition between dopamine and 6RBPH4 on the TH molecule.

Acknowledgment: This work was supported by grants-in- aid from the Ministry of Education, Science, Sports, and Cul- ture of Japan to A.O. and also by grants-in-aid from Fujita Health University, Japan, to A.N., A.O., and T.N. We thank Ms. Mina Tsutsui and Ms. Sand Kawarazaki for technical assistance.

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