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Identification of the Essential Cysteinyl Residue Located in the Active Site of Human PhenylethanolamineN-Methyltransferase
BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS ARTICLE NO.
249, 405–409 (1998)
RC989143
Identification of the Essential Cysteinyl Residue Located in the Active Site of Human Phenylethanolamine N-Methyltransferase Norio Kaneda,*,†,1 Kiyomi Hikita,* Yoriko Naruse,* Taeko Fukuo,* Kenichiro Matsubara,† and Toshiharu Nagatsu‡ *Department of Analytical Neurobiology, Faculty of Pharmacy, Meijo University, Nagoya 468-8503, Japan; †Department of Biochemistry, Nagoya University School of Medicine, Nagoya 466-8550, Japan; and ‡Division of Molecular Genetics II (Neurochemistry), Institute for Comprehensive Medical Science, School of Medicine, Fujita Health University, Toyoake, Aichi 470-1192, Japan
Received July 9, 1998
Phenylethanolamine N-methyltransferase (PNMT) catalyzes the production of epinephrine from norepinephrine using S-adenosyl-L-methionine as a methyl donor. Previous studies of chemical modification of the PNMT with reagents specific to Cys residues showed that the enzyme contains a Cys residue essential for its activity. Each of the six Cys residues in human PNMT was changed to Ser by PCR-based site-directed mutagenesis, and each mutant PNMT was expressed in Escherichia coli to identify the functionally important Cys residue. The six mutants (C48S, C60S, C91S, C131S, C139S, and C183S) and the wild-type enzyme were expressed at almost the same levels as revealed by Western blotting analysis. Kinetic parameters (apparent Km and Vmax) of C48S, C60S, C91S, C131S, and C139S for the substrates, norepinephrine and Sadenosyl-L-methionine, showed similar values to those of the wild-type enzyme. However, C183S exhibited markedly reduced enzyme activity with less than 3% of the wild-type Vmax and with ca. sixfold increased apparent Km values for both substrates. These results suggested that Cys183 plays an important role in the activity of human PNMT. q 1998 Academic Press
Phenylethanolamine N-methyltransferase (PNMT) catalyzes the formation of epinephrine from norepinephrine, the last step of the catecholamine biosynthesis pathway (1, 2). PNMT has been purified from the adrenal medulla of cattle and many other mammals, and has been characterized in detail (3-7). PNMT is a monomeric enzyme with M.W. 31,000. Similarly to 1 To whom correspondence should be addressed. Fax: /81-52-8348090. E-mail: [email protected].
other small molecular N-methyltransferases, S-adenosyl-L-methionine is required for the enzyme reaction as a methyl donor. In addition to its major role in producing adrenomedullary hormones, PNMT has some important roles in blood pressure and neuroendocrine regulation in the central nervous system (8, 9). Therefore, information regarding the structure of active site may be useful for development of selective and potent inhibitors for possible use as antihypertensive agents (10). Grunewald et al. (11) developed a theoretical active site model of PNMT based on the structures of known PNMT ligands and inhibitors. They proposed that the active site is composed of a polar amine binding area and two lipophilic pockets. However, particular amino acid residues involved in the active site have not been reported. Previous studies using chemical reagents specific to Cys residues such as p-chloromercuribenzoate (PCMB) suggested that PNMT contains a Cys residue essential for the catalytic activity (2, 3). We previously isolated human PNMT cDNA, and demonstrated that the human enzyme has six Cys residues at positions 48, 60, 91, 131, 139 and 183 in its primary amino acid sequence (12). Based on these findings, we attempted to identify the essential Cys residue of the enzyme using PCR-based site-directed mutagenesis. We changed each Cys residue to Ser, and produced the mutants in Escherichia coli. Here, we will discuss identification of the essential Cys residue located in the active site of human PNMT. MATERIALS AND METHODS Vector construction for expression in E. coli. The coding region of human PNMT cDNA was isolated from the full-length cDNA clone phPNMT (12) using PCR and specific primers with appropriate restriction sites. The PCR product was digested with corresponding 0006-291X/98 $25.00
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restriction enzymes, and subcloned into the bacterial expression vector pKK223-3 (Amersham Pharmacia Biotech, Uppsala, Sweden). In a preliminary study to check expression efficiency, we found that modification of the sequence preceding the initial Met markedly improved expression. The modified sequence was as follows: 5*-GGAGGAAAAAATTATG-3*. The Shine-Dalgarno sequence and the initial Met codon are underlined. Site-directed mutagenesis. Site-directed mutagenesis of Cys codons, TGC, into Ser codons, TCT, was performed according to the method of Ito et al. (13). First, the coding region of human PNMT cDNA was subcloned into pBluescript KS (/) vector (Stratagene, La Jolla, CA). Nucleotide sequences of primers I, II and III were as follows: primer I (sense), 5*-AGCTCGGAATTAACCCTCACTAAAG-3*; primer II (antisense), 5*-GCAAGCTTAGGTGCCACTTCAAAG-3*; primer III (sense), 5*-AGCTTGATATCTAATTCCGGAGG-3*. The mutated nucleotide in the Eco RI site is underlined. Nucleotide sequences of primer IV (antisense) used for introduction of the mutation were as follows: primer IV48, 5*-CCGTTCGGGTTAGACAGGTCCCC-3*; primer IV60, 5*-GTCTGCGCCAAAGAGCGCAGCTTCC-3*; primer IV91, 5*-GCTAGAGGCACTGAGCAGCTGGTACAC-3*; primer IV131, 5*-CTTGCCCTCAATGAGAGAGGCATGTTGG-3*; primer IV139, 5*-CTTATCCTGCCAAGATTCCCCCTTGCCCTC-3*; and primer IV183, 5*-GCTCACAGCCTCCAAAGAGAAGGCAGAGACCA-3*. Mutated nucleotides are underlined. The nucleotide sequence of the entire coding region was confirmed by the cycle dye terminator method using an automatic fluorescence DNA sequencer, Genetic Analyzer 310-2 (Perkin-Elmer Applied Biosystems, Foster City, CA). Expression of wild-type and mutant PNMTs in E. coli. Wild-type and mutant PNMT cDNA were subcloned into the expression vector pKK223-3 at Eco RI and Hind III sites, and introduced into E. coli strain JM109. Expression of recombinant proteins was carried out as described (14). Briefly, each bacterial clone was first incubated at 377C with vigorous shaking in LB medium supplemented with ampicillin (100 mg/ml). After incubation for ca. 3 h (OD600 Å ca. 1.0), 1 mM isopropyl-b-D-thiogalactopyranoside (IPTG) was added to the medium to induce synthesis of the desired protein, and the bacteria were cultured for a further 4 h. After centrifugation at 5,000g for 5 min, the bacterial pellet was washed with a solution of 0.1 mM Tris HCl (pH 7.5) and 0.1 M NaCl, suspended with a solution of 30 mM Tris HCl, 1 mM dithiothreitol and 1 mM EDTA, and then homogenized twice for 30 sec each time using an ultrasonic homogenizer (SMT, Japan). The homogenate was centrifuged at 543,000g for 15 min at 47C in a TL-100 ultracentrifuge (Beckman, Palo Alto, CA), and the resulting supernatant was used as the enzyme solution. Electrophoresis and Western blotting analysis. SDS-PAGE was performed according to the method of Laemmli et al. (15) using 12.5% polyacrylamide gels, and was followed by Western blotting analysis as reported previously (16). After electrophoresis, proteins were transferred onto polyvinylidene difluoride membranes (Millipore, Bedford, MA), and incubated with rabbit anti-bovine PNMT antiserum (1:250 dilution). After washing and incubation with horseradish peroxidase-conjugated anti-rabbit IgG (1:2,000 dilution, Bio-Rad, Hercules, CA), immunoreactive bands were visualized with 4-chloro1-naphthol and 0.015% H2O2. Assay of PNMT activity. PNMT activity was assayed by a modification of the method reported previously (17) using HPLC with electrochemical detection; in the present study, monoamine oxidase inhibitor was omitted from the reaction mixture (250 ml). Enzyme reaction was performed at 377C for 1 hr, followed by addition of perchloric acid solution (final conc. 0.4 M) containing 250 pmol of dihydroxybenzenzylamine (DHBA) as an internal standard, 0.5% Na2S2O5 and 0.25% EDTA. Norepinephrine, epinephrine and DHBA were separated within 12 min on a Chemcosorb ODS column (4.6 f x 150 mm, Chemco, Osaka, Japan), using 0.1 M potassium phosphate buffer (pH 3.5) containing 30 mg/l sodium octanesulfonate and 7.4 mg/l EDTA as the mobile phase.
FIG. 1. Expression of wild-type and mutant PNMTs in E. coli. The supernatant (20 mg protein) from each bacterial clone for the wild-type and the mutant PNMT was analyzed by 12.5% PAGE, followed by Western blotting using specific anti-bovine PNMT antiserum. Molecular weights of standard proteins are indicated on the left.
RESULTS Expression of wild-type and mutant human PNMT in E. coli. Wild-type and mutant PNMT cDNA expression vectors were introduced into E. coli, and expression was induced by adding 1 mM IPTG. The level of expression of each mutant protein was examined by Western blotting analysis using the crude extract which was prepared 4 h after IPTG induction. As shown in Fig. 1, wild-type and all the six mutants expressed PNMT protein almost at the same level. No enzyme activity was detected in JM109 which was transformed with pKK223-3 vector alone (data not shown). Accordingly, the following kinetic studies were performed using crude extract as the enzyme solution. Identification of the essential Cys residue in the human PNMT. Figure 2 shows substrate concentrationdependency of wild-type and mutant PNMTs. As shown in Fig. 2a, alteration of Cys residues at positions 48, 60, 91, 131 and 139 to Ser showed no marked effect on the norepinephrine concentration-dependency of enzyme activity compared with the wild-type enzyme. In contrast, C183S in which the Cys residue at position 183 was replaced with Ser showed a marked decrease in activity compared with the wild-type enzyme. Similar concentration-dependency was observed in the case of S-adenosyl-L-methionine, another substrate of the enzyme (Fig. 2b): C183S exclusively exhibited a marked decrease in enzyme activity, whereas none of the other mutants showed any marked changes compared with the wild-type enzyme. To obtain kinetic parameters, Lineweaver–Burk
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FIG. 2. Enzyme kinetics of the wild-type and mutant PNMTs. (a) Enzyme activities were measured at various concentrations of norepinephrine and 18 mM S-adenosyl-L-methionine. (b) Enzyme activities were measured at various concentrations of S-adenosyl-Lmethionine and 16 mM norepinephrine. l, wild-type; m, C48S; j, C60S; s, C91S; n, C131S; h, C139S; ., C183S.
plots were generated for both substrates. The apparent Km and Vmax values are summarized in Table 1 and Table 2. As shown in Table 1, the apparent Km value of the wild-type enzyme for norepinephrine was 18.4 mM. Those of mutants C48S, C60S, C91S, C131S and C139S ranged from 16 to 20 mM, and these values were almost the same as that for the wild-type enzyme. These all mutants showed essentially the same Vmax, indicating that these residues had no fundamental effect on enzyme activity. In contrast, C183S showed an apparent Km value of 110 mM for norepinephrine, ca. 6-fold larger than that of the wild-type, and the Vmax value was reduced to only 2.7% of that of the wild-type. Almost the same results were obtained with kinetic parameters for S-adenosyl-L-methionine (Table 2). Only C183S showed a ca. 6-fold larger value of appar-
A previous study with a chemical reagent specific to SH groups, p-hydroxymercuribenzoate, suggested that bovine PNMT has 8.5 -SH groups per mol of the enzyme (3). cDNA cloning of bovine and human PNMT revealed that both enzymes possess six Cys residues in their primary structures (12, 18). Therefore, these Cys residues were all considered to exist in the free -SH form, and not in disulfide bridges. In addition, a previous
TABLE 1
TABLE 2
Kinetic Parameters of Wild-Type and Mutant PNMTs for Norepinephrine
Kinetic Parameters of Wild-Type and Mutant PNMTs for S-Adenosyl-L-methionine
Wild-type C48S C60S C91S C131S C139S C183S
Km (mM)
Vmax (nmol/h/mg protein)
Vmax % Vmax wild
18.4 16.6 20.1 21.6 18.4 19.5 110
147 150 137 144 147 148 4.0
100 103 94 99 100 101 2.7
ent Km, and only 2.6% of Vmax value compared with the wild-type enzyme. These results strongly suggest that the Cys residue at position 183 is the most important among the six Cys residues for enzyme activity. DISCUSSION
Wild-type C48S C60S C91S C131S C139S C183S
Km (mM)
Vmax (nmol/h/mg protein)
Vmax % Vmax wild
19.4 24.7 15.2 21.6 25.7 27.5 118
186 211 137 178 216 230 4.9
100 113 74 96 116 124 2.6
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FIG. 3. Amino acid sequences of human, bovine and mouse PNMT. Amino acid sequences are aligned to show the maximum homology. Conserved residues are shown by open boxes with Cys residues highlighted by solid boxes.
study showed that modification with PCMB inactivated the enzyme, suggesting that PNMT has at least one -SH group which is essential for the activity. Human PNMT has six Cys residues at positions 48, 60, 91, 131, 139 and 183. To identify this essential Cys residue, we constructed cDNAs encoding wild-type and mutant human PNMT in which each of the six Cys residues was changed to Ser by PCR-based site-directed mutagenesis. These molecules were then produced in an E. coli expression system. Western blotting analysis showed that bacteria bearing constructs for each mutant as well as wild-type expressed almost the same amounts of the enzyme protein in a soluble form. Of these mutant PNMTs, C183S exclusively showed reduced enzyme activity. In C183S, the apparent Km values for norepinephrine or S-adenosyl-L-methionine were both increased by ca. 6-fold, and Vmax was reduced to less than 3% of the wild-type enzyme. These results suggested that Cys183 reacted with PCMB to inactivate the enzyme. Chemical modification with PCMB resulted in complete loss of the enzyme activity (2, 3). However, the change from Cys to Ser at position 183 did not lead to complete loss of activity. This discrepancy may be attributed to the bulkiness of PCMB, which may interfere with substrate binding to the enzyme through steric hindrance. In contrast, the effect of alteration of the -SH to -OH group would be minimal in terms of steric hindrance because these groups are similar in size and therefore the conformation of the entire enzyme would remain unchanged. As Vmax was
markedly reduced in C183S, Cys183 was suggested to be one of the essential residues residing at the catalytic center of the enzyme. This residue may be involved in substrate binding as suggested by the increased Km values for the substrates. Determination of the threedimensional structure of the enzyme will help in understanding the details of the catalytic center of PNMT. To estimate the importance of each Cys residue, the amino acid sequences of human, bovine and mouse PNMT were aligned. As shown in Fig. 3, Cys60, Cys91, Cys131 and Cys183 were conserved between these three species suggesting the structural and/or functional importance of these residues. Interestingly, in bovine and mouse homologues, amino acids at the position corresponding to Cys48 and Cys139 are replaced with Ser, and these were considered to be naturally occurring site-directed mutations. Therefore, the finding that C48S and C139S did not show significantly different enzyme activities compared to the wild-type enzyme seems reasonable. Of the remaining four conserved Cys residues at positions 60, 91, 131 and 183, Cys183 is located at the most conserved region in the primary structure. From these results, it could be concluded that the active site of the enzyme is in the C-terminal half, and that Cys183 is one of the most important residues involved in the active site of the enzyme. ACKNOWLEDGMENTS This work was supported by grants from the Ministry of Education, Science, and Culture of Japan; from the Science Research Promotion
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Fund of the Promotion and Mutual Aid Corporation for Private Schools of Japan; and from the Fugaku Trust for Medicinal Research. 9.
REFERENCES 1. Kirshner, N., and Goodall, M. (1957) Biochim. Biophys. Acta 24, 658–659. 2. Axelrod, J. (1962) J. Biol. Chem. 237, 1656–1660. 3. Connett, R. J., and Kirshner, N. (1970) J. Biol. Chem. 245, 329– 334. 4. Pohorecky, L. A., and Baliga, B. S. (1973) Arch. Biochem. Biophys. 156, 703–711. 5. Wong, D. L., Yamasaki, L., and Ciaranello, R. D. (1987) Brain Res. 410, 32–44. 6. Hurst, J. H., Guchhait, R. B., Billigsley, M. L., Stolk, J. M., and Lovenberg, W. (1983) Biochem. Biophys. Res. Commun. 112, 1061–1068.
10. 11.
12.
13. 14.
15. 16.
7. Fuller, R. W. (1987) Methods Enzymol. 142, 655–660. 8. Fuxe, K., Ganten, D., Bolme, P., Agnati, L. F. Ho¨kfelt, T., Andersson, K., Goldstein, M., Ha¨rfstrand, A., Unger T., and Rascher, W. (1980) in Central Adrenaline Neurons: Basic Aspects and Their Role in Cardiovascular Disease (Fuxe, K.,
17. 18.
Goldstein, M., Ho¨kfelt, B., and Ho¨kfelt, T., Eds.), pp. 259–276, Pergamon Press, New York. Terry, L. C. (1988) in Epinephrine in the Central Nervous System (Stolk, J. M., U’Prichard, D. C., and Fuxe, K., Eds.), pp. 154– 165, Oxford Univ. Press, New York. Fuller, R. W. (1982) Annu. Rev. Pharmacol. Toxicol. 22, 31–55. Grunewald, G. L., Monn, J. A., and Sall, D. J. (1988) in Epinephrine in the Central Nervous System (Stolk, J. M., U’Prichard, D. C., and Fuxe, K., Eds.), pp. 117–140, Oxford Univ. Press, New York. Kaneda, N., Ichinose, H., Kabayashi, K., Oka, K., Kishi, F., Nakazawa, A., Kurosawa, Y., Fujita, K., and Nagatsu, T. (1988) J. Biol. Chem. 263, 7672–7677. Ito, W., Ishiguro, H., and Kurosawa, Y. (1991) Gene 102, 67–70. Sambrook, J., Fritsch, E. F., and Maniatis, T. (1989) in Molecular Cloning: A Laboratory Manual, pp. 17.2–17.44, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY. Laemmli, U. K. (1970) Nature 227, 680–685. Towbin, H., Staehelin, T., and Gordon, J. (1979) Proc. Natl. Acad. Sci. USA 76, 4350–4354. Trocewicz, J., Oka, K., and Nagatsu, T. (1982) J. Chromatogr. 227, 407–413. Baetge, E. E., Suh, Y. H., and Joh, T. H. (1986) Proc. Natl. Acad. Sci. USA 83, 5454–5458.
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RC989143
Identification of the Essential Cysteinyl Residue Located in the Active Site of Human Phenylethanolamine N-Methyltransferase Norio Kaneda,*,†,1 Kiyomi Hikita,* Yoriko Naruse,* Taeko Fukuo,* Kenichiro Matsubara,† and Toshiharu Nagatsu‡ *Department of Analytical Neurobiology, Faculty of Pharmacy, Meijo University, Nagoya 468-8503, Japan; †Department of Biochemistry, Nagoya University School of Medicine, Nagoya 466-8550, Japan; and ‡Division of Molecular Genetics II (Neurochemistry), Institute for Comprehensive Medical Science, School of Medicine, Fujita Health University, Toyoake, Aichi 470-1192, Japan
Received July 9, 1998
Phenylethanolamine N-methyltransferase (PNMT) catalyzes the production of epinephrine from norepinephrine using S-adenosyl-L-methionine as a methyl donor. Previous studies of chemical modification of the PNMT with reagents specific to Cys residues showed that the enzyme contains a Cys residue essential for its activity. Each of the six Cys residues in human PNMT was changed to Ser by PCR-based site-directed mutagenesis, and each mutant PNMT was expressed in Escherichia coli to identify the functionally important Cys residue. The six mutants (C48S, C60S, C91S, C131S, C139S, and C183S) and the wild-type enzyme were expressed at almost the same levels as revealed by Western blotting analysis. Kinetic parameters (apparent Km and Vmax) of C48S, C60S, C91S, C131S, and C139S for the substrates, norepinephrine and Sadenosyl-L-methionine, showed similar values to those of the wild-type enzyme. However, C183S exhibited markedly reduced enzyme activity with less than 3% of the wild-type Vmax and with ca. sixfold increased apparent Km values for both substrates. These results suggested that Cys183 plays an important role in the activity of human PNMT. q 1998 Academic Press
Phenylethanolamine N-methyltransferase (PNMT) catalyzes the formation of epinephrine from norepinephrine, the last step of the catecholamine biosynthesis pathway (1, 2). PNMT has been purified from the adrenal medulla of cattle and many other mammals, and has been characterized in detail (3-7). PNMT is a monomeric enzyme with M.W. 31,000. Similarly to 1 To whom correspondence should be addressed. Fax: /81-52-8348090. E-mail: [email protected].
other small molecular N-methyltransferases, S-adenosyl-L-methionine is required for the enzyme reaction as a methyl donor. In addition to its major role in producing adrenomedullary hormones, PNMT has some important roles in blood pressure and neuroendocrine regulation in the central nervous system (8, 9). Therefore, information regarding the structure of active site may be useful for development of selective and potent inhibitors for possible use as antihypertensive agents (10). Grunewald et al. (11) developed a theoretical active site model of PNMT based on the structures of known PNMT ligands and inhibitors. They proposed that the active site is composed of a polar amine binding area and two lipophilic pockets. However, particular amino acid residues involved in the active site have not been reported. Previous studies using chemical reagents specific to Cys residues such as p-chloromercuribenzoate (PCMB) suggested that PNMT contains a Cys residue essential for the catalytic activity (2, 3). We previously isolated human PNMT cDNA, and demonstrated that the human enzyme has six Cys residues at positions 48, 60, 91, 131, 139 and 183 in its primary amino acid sequence (12). Based on these findings, we attempted to identify the essential Cys residue of the enzyme using PCR-based site-directed mutagenesis. We changed each Cys residue to Ser, and produced the mutants in Escherichia coli. Here, we will discuss identification of the essential Cys residue located in the active site of human PNMT. MATERIALS AND METHODS Vector construction for expression in E. coli. The coding region of human PNMT cDNA was isolated from the full-length cDNA clone phPNMT (12) using PCR and specific primers with appropriate restriction sites. The PCR product was digested with corresponding 0006-291X/98 $25.00
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restriction enzymes, and subcloned into the bacterial expression vector pKK223-3 (Amersham Pharmacia Biotech, Uppsala, Sweden). In a preliminary study to check expression efficiency, we found that modification of the sequence preceding the initial Met markedly improved expression. The modified sequence was as follows: 5*-GGAGGAAAAAATTATG-3*. The Shine-Dalgarno sequence and the initial Met codon are underlined. Site-directed mutagenesis. Site-directed mutagenesis of Cys codons, TGC, into Ser codons, TCT, was performed according to the method of Ito et al. (13). First, the coding region of human PNMT cDNA was subcloned into pBluescript KS (/) vector (Stratagene, La Jolla, CA). Nucleotide sequences of primers I, II and III were as follows: primer I (sense), 5*-AGCTCGGAATTAACCCTCACTAAAG-3*; primer II (antisense), 5*-GCAAGCTTAGGTGCCACTTCAAAG-3*; primer III (sense), 5*-AGCTTGATATCTAATTCCGGAGG-3*. The mutated nucleotide in the Eco RI site is underlined. Nucleotide sequences of primer IV (antisense) used for introduction of the mutation were as follows: primer IV48, 5*-CCGTTCGGGTTAGACAGGTCCCC-3*; primer IV60, 5*-GTCTGCGCCAAAGAGCGCAGCTTCC-3*; primer IV91, 5*-GCTAGAGGCACTGAGCAGCTGGTACAC-3*; primer IV131, 5*-CTTGCCCTCAATGAGAGAGGCATGTTGG-3*; primer IV139, 5*-CTTATCCTGCCAAGATTCCCCCTTGCCCTC-3*; and primer IV183, 5*-GCTCACAGCCTCCAAAGAGAAGGCAGAGACCA-3*. Mutated nucleotides are underlined. The nucleotide sequence of the entire coding region was confirmed by the cycle dye terminator method using an automatic fluorescence DNA sequencer, Genetic Analyzer 310-2 (Perkin-Elmer Applied Biosystems, Foster City, CA). Expression of wild-type and mutant PNMTs in E. coli. Wild-type and mutant PNMT cDNA were subcloned into the expression vector pKK223-3 at Eco RI and Hind III sites, and introduced into E. coli strain JM109. Expression of recombinant proteins was carried out as described (14). Briefly, each bacterial clone was first incubated at 377C with vigorous shaking in LB medium supplemented with ampicillin (100 mg/ml). After incubation for ca. 3 h (OD600 Å ca. 1.0), 1 mM isopropyl-b-D-thiogalactopyranoside (IPTG) was added to the medium to induce synthesis of the desired protein, and the bacteria were cultured for a further 4 h. After centrifugation at 5,000g for 5 min, the bacterial pellet was washed with a solution of 0.1 mM Tris HCl (pH 7.5) and 0.1 M NaCl, suspended with a solution of 30 mM Tris HCl, 1 mM dithiothreitol and 1 mM EDTA, and then homogenized twice for 30 sec each time using an ultrasonic homogenizer (SMT, Japan). The homogenate was centrifuged at 543,000g for 15 min at 47C in a TL-100 ultracentrifuge (Beckman, Palo Alto, CA), and the resulting supernatant was used as the enzyme solution. Electrophoresis and Western blotting analysis. SDS-PAGE was performed according to the method of Laemmli et al. (15) using 12.5% polyacrylamide gels, and was followed by Western blotting analysis as reported previously (16). After electrophoresis, proteins were transferred onto polyvinylidene difluoride membranes (Millipore, Bedford, MA), and incubated with rabbit anti-bovine PNMT antiserum (1:250 dilution). After washing and incubation with horseradish peroxidase-conjugated anti-rabbit IgG (1:2,000 dilution, Bio-Rad, Hercules, CA), immunoreactive bands were visualized with 4-chloro1-naphthol and 0.015% H2O2. Assay of PNMT activity. PNMT activity was assayed by a modification of the method reported previously (17) using HPLC with electrochemical detection; in the present study, monoamine oxidase inhibitor was omitted from the reaction mixture (250 ml). Enzyme reaction was performed at 377C for 1 hr, followed by addition of perchloric acid solution (final conc. 0.4 M) containing 250 pmol of dihydroxybenzenzylamine (DHBA) as an internal standard, 0.5% Na2S2O5 and 0.25% EDTA. Norepinephrine, epinephrine and DHBA were separated within 12 min on a Chemcosorb ODS column (4.6 f x 150 mm, Chemco, Osaka, Japan), using 0.1 M potassium phosphate buffer (pH 3.5) containing 30 mg/l sodium octanesulfonate and 7.4 mg/l EDTA as the mobile phase.
FIG. 1. Expression of wild-type and mutant PNMTs in E. coli. The supernatant (20 mg protein) from each bacterial clone for the wild-type and the mutant PNMT was analyzed by 12.5% PAGE, followed by Western blotting using specific anti-bovine PNMT antiserum. Molecular weights of standard proteins are indicated on the left.
RESULTS Expression of wild-type and mutant human PNMT in E. coli. Wild-type and mutant PNMT cDNA expression vectors were introduced into E. coli, and expression was induced by adding 1 mM IPTG. The level of expression of each mutant protein was examined by Western blotting analysis using the crude extract which was prepared 4 h after IPTG induction. As shown in Fig. 1, wild-type and all the six mutants expressed PNMT protein almost at the same level. No enzyme activity was detected in JM109 which was transformed with pKK223-3 vector alone (data not shown). Accordingly, the following kinetic studies were performed using crude extract as the enzyme solution. Identification of the essential Cys residue in the human PNMT. Figure 2 shows substrate concentrationdependency of wild-type and mutant PNMTs. As shown in Fig. 2a, alteration of Cys residues at positions 48, 60, 91, 131 and 139 to Ser showed no marked effect on the norepinephrine concentration-dependency of enzyme activity compared with the wild-type enzyme. In contrast, C183S in which the Cys residue at position 183 was replaced with Ser showed a marked decrease in activity compared with the wild-type enzyme. Similar concentration-dependency was observed in the case of S-adenosyl-L-methionine, another substrate of the enzyme (Fig. 2b): C183S exclusively exhibited a marked decrease in enzyme activity, whereas none of the other mutants showed any marked changes compared with the wild-type enzyme. To obtain kinetic parameters, Lineweaver–Burk
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FIG. 2. Enzyme kinetics of the wild-type and mutant PNMTs. (a) Enzyme activities were measured at various concentrations of norepinephrine and 18 mM S-adenosyl-L-methionine. (b) Enzyme activities were measured at various concentrations of S-adenosyl-Lmethionine and 16 mM norepinephrine. l, wild-type; m, C48S; j, C60S; s, C91S; n, C131S; h, C139S; ., C183S.
plots were generated for both substrates. The apparent Km and Vmax values are summarized in Table 1 and Table 2. As shown in Table 1, the apparent Km value of the wild-type enzyme for norepinephrine was 18.4 mM. Those of mutants C48S, C60S, C91S, C131S and C139S ranged from 16 to 20 mM, and these values were almost the same as that for the wild-type enzyme. These all mutants showed essentially the same Vmax, indicating that these residues had no fundamental effect on enzyme activity. In contrast, C183S showed an apparent Km value of 110 mM for norepinephrine, ca. 6-fold larger than that of the wild-type, and the Vmax value was reduced to only 2.7% of that of the wild-type. Almost the same results were obtained with kinetic parameters for S-adenosyl-L-methionine (Table 2). Only C183S showed a ca. 6-fold larger value of appar-
A previous study with a chemical reagent specific to SH groups, p-hydroxymercuribenzoate, suggested that bovine PNMT has 8.5 -SH groups per mol of the enzyme (3). cDNA cloning of bovine and human PNMT revealed that both enzymes possess six Cys residues in their primary structures (12, 18). Therefore, these Cys residues were all considered to exist in the free -SH form, and not in disulfide bridges. In addition, a previous
TABLE 1
TABLE 2
Kinetic Parameters of Wild-Type and Mutant PNMTs for Norepinephrine
Kinetic Parameters of Wild-Type and Mutant PNMTs for S-Adenosyl-L-methionine
Wild-type C48S C60S C91S C131S C139S C183S
Km (mM)
Vmax (nmol/h/mg protein)
Vmax % Vmax wild
18.4 16.6 20.1 21.6 18.4 19.5 110
147 150 137 144 147 148 4.0
100 103 94 99 100 101 2.7
ent Km, and only 2.6% of Vmax value compared with the wild-type enzyme. These results strongly suggest that the Cys residue at position 183 is the most important among the six Cys residues for enzyme activity. DISCUSSION
Wild-type C48S C60S C91S C131S C139S C183S
Km (mM)
Vmax (nmol/h/mg protein)
Vmax % Vmax wild
19.4 24.7 15.2 21.6 25.7 27.5 118
186 211 137 178 216 230 4.9
100 113 74 96 116 124 2.6
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FIG. 3. Amino acid sequences of human, bovine and mouse PNMT. Amino acid sequences are aligned to show the maximum homology. Conserved residues are shown by open boxes with Cys residues highlighted by solid boxes.
study showed that modification with PCMB inactivated the enzyme, suggesting that PNMT has at least one -SH group which is essential for the activity. Human PNMT has six Cys residues at positions 48, 60, 91, 131, 139 and 183. To identify this essential Cys residue, we constructed cDNAs encoding wild-type and mutant human PNMT in which each of the six Cys residues was changed to Ser by PCR-based site-directed mutagenesis. These molecules were then produced in an E. coli expression system. Western blotting analysis showed that bacteria bearing constructs for each mutant as well as wild-type expressed almost the same amounts of the enzyme protein in a soluble form. Of these mutant PNMTs, C183S exclusively showed reduced enzyme activity. In C183S, the apparent Km values for norepinephrine or S-adenosyl-L-methionine were both increased by ca. 6-fold, and Vmax was reduced to less than 3% of the wild-type enzyme. These results suggested that Cys183 reacted with PCMB to inactivate the enzyme. Chemical modification with PCMB resulted in complete loss of the enzyme activity (2, 3). However, the change from Cys to Ser at position 183 did not lead to complete loss of activity. This discrepancy may be attributed to the bulkiness of PCMB, which may interfere with substrate binding to the enzyme through steric hindrance. In contrast, the effect of alteration of the -SH to -OH group would be minimal in terms of steric hindrance because these groups are similar in size and therefore the conformation of the entire enzyme would remain unchanged. As Vmax was
markedly reduced in C183S, Cys183 was suggested to be one of the essential residues residing at the catalytic center of the enzyme. This residue may be involved in substrate binding as suggested by the increased Km values for the substrates. Determination of the threedimensional structure of the enzyme will help in understanding the details of the catalytic center of PNMT. To estimate the importance of each Cys residue, the amino acid sequences of human, bovine and mouse PNMT were aligned. As shown in Fig. 3, Cys60, Cys91, Cys131 and Cys183 were conserved between these three species suggesting the structural and/or functional importance of these residues. Interestingly, in bovine and mouse homologues, amino acids at the position corresponding to Cys48 and Cys139 are replaced with Ser, and these were considered to be naturally occurring site-directed mutations. Therefore, the finding that C48S and C139S did not show significantly different enzyme activities compared to the wild-type enzyme seems reasonable. Of the remaining four conserved Cys residues at positions 60, 91, 131 and 183, Cys183 is located at the most conserved region in the primary structure. From these results, it could be concluded that the active site of the enzyme is in the C-terminal half, and that Cys183 is one of the most important residues involved in the active site of the enzyme. ACKNOWLEDGMENTS This work was supported by grants from the Ministry of Education, Science, and Culture of Japan; from the Science Research Promotion
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Fund of the Promotion and Mutual Aid Corporation for Private Schools of Japan; and from the Fugaku Trust for Medicinal Research. 9.
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