Vol. 173, No. 2, 1990
BIOCHEMICAL AND BIOPHYSICALRESEARCH COMMUNICATIONS Pages 756-762
December 14, 1990
ROLE OF TRYPTOPHAN 248 OF TRYPTOPHANASE FROM
IN THE ACTIVE SITE ESCHERICHIA COLI 1
Yasushi Kawata 2, Nobuharu Tsujimoto, Shunsuke Tani, Tomohiro Mizobata, and Masanobu Tokushige 3 Department of Chemistry, Faculty of Science, Kyoto University, Kyoto 606, Japan Received November 8, 1990
Tryptophan 248, located in the active site of tryptophanase from Escherichia coli, has been replaced with phenylalanine by site-directed mutagenesis. Judging from CD and fluorescence spectra, the global structure of the mutant enzyme was found to be the same as that of the wild-type enzyme. The binding affinity of the mutant enzyme for the coenzyme pyridoxal Y-phosphate (PLP) was reduced tenfold compared to the wild-type enzyme. Kinetic analyses under PLP-saturated conditions indicated that the Km values of the mutant enzyme for substrates are the same as those of wild-type enzyme but the kcat values are decreased to about 85%, which accounts for the overall activity decrease. These findings suggest that tryptophan 248 interacts closely with PLP and plays an important role in the catalytic reaction. ~ 199oAoademioP..... ~no.
Tryptophanase (L-tryptophan indole-lyase, EC 4.1.99.1) catalyses the reversible degradation of tryptophan to indole, pyruvate, and ammonia (1). The enzyme from E. coli consists of four identical subunits with a molecular weight of 52,242 (total molecular weight 208,968), as determined by DNA sequence analysis (2, 3). Each subunit binds one pyridoxal 5'-phosphate (PLP) molecule
1This work was supported in part by Grants-in-Aid for Scientific Research (No.01780198) from the Ministry of Education, Science and Culture of Japan and also by Amano Pharmaceutical Co. Foundation. 2To whom correspondence should be addressed. 3Deceased October 4, 1990. Abbreviations: CD, circular dichroism; PLP, pyridoxal 5'-phosphate; SOPC, S-o-nitrophenyl-L-cysteine; W248F, mutant tryptophanase in which tryptophan 248 has been replaced with phenylalanine. 0006-291X/90 $1.50 Copyright © 1990 by Academic Press, Inc. All rights of reproduction in any form reserved.
756
Vol. 173, No. 2, 1990
BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS
to its active site as a coenzyme. Tryptophanase possesses two tryptophans in its amino acid sequence. One of these, Trp 248, has been previously reported to be located in the active site region (4). In order to study the role of this tryptophan residue in the catalytic mechanism, we have prepared a mutant enzyme by sitedirected mutagenesis, in which Trp 248 was replaced with phenylalanine (W248F). From the measurements of PLP-binding affinity and kinetic analyses of enzymatic action, it was indicated that Trp 248 interacts closely with the PLP and plays an important role in the catalytic reaction. MATERIALS AND METHODS
Chemicals. L-tryptophan, NADH, PLP, and lactate dehydrogenase from hog muscle were purchased from nacalai tesque. S-o-nitrophenyl-L-cysteine (SOPC) was prepared by the reaction of L-cysteine hydrochloride hydrate with 2fluoronitrobenzene in dimethylformamide containing triethylamine (5). Aminohexyl-Sepharose was obtained from Pharmacia (Uppsala, Sweden). All other chemicals were of the highest grade commercially available. Preparation of W248F mutant. A 220-base pair PstI/KpnI fragment was subcloned from pMD6B which encodes the E. coli B/lt7-A tryptophanase (2) into M13mpl9, and single-stranded DNA was prepared. Oligonucleotide directed sitespecific mutagenesis was performed by the method of Eckstein (6) using a kit from Amersham Corp. The mutagenic oligonucleotide (5'-CTC GAT GGT GAA GTC TTT GTA-3') was synthesized using an Applied Biosystems DNA synthesizer (model 381A). Candidate plaques were screened for the mutation by digestion of the plasmid with AvalI (the mutation lacks the restriction site), and the mutated DNA fragment was sequenced by the dideoxy method (7). Doublestranded RF DNA was prepared from the mutant phage and the 220-base pair PstI/KpnI fragment was isolated and ligated at the original PstI/KpnI site of pMD6B. To insure that the desired mutation had been introduced, the resulting plasmid (pMD6BW248F) was sequenced again in the vicinity of the mutation and the ligation sites by subcloning of a 627-base pair HhaI fragment to M13mp19. The pMD6BW248F was then transformed into E. coli K-12 MD55, which is an auxotroph of tryptophan. The cells were grown and the enzyme was purified as described previously (8). In the present experiments, wild-type and W248F were further purified by using an affinity column of Sepharose-bound PLP-alanine complex, SP-C-Ala. SP-C-Ala was prepared by the method of Ikeda et al. (9) with modifications. The enzyme (20 mg) was applied to the column (0.5 x 10 cm) previously equilibrated with 10 mM potassium phosphate buffer, pH 7.0 and washed thoroughly with 1 M potassium phosphate buffer, pH 7.0, and then eluted with 50 mM potassium acetate, pH 5.0, containing 50 ktM PLP. The buffer of the eluted enzyme was immediately substituted with 20 mM potassium phosphate buffer, pH 7.0, containing 0.5 mM PLP and 5 mM dithiothreitol. The specific activity of the enzyme obtained thus was improved. Enzyme assays. Routine enzyme assays were performed with 0.33 mM SOPC in 0.1 M potassium phosphate buffer, pH 7.8, at 30°C, measured at 370 n m ( e = 757
Vol. 173, No. 2, 1990
BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS
1.86 x 103 M-lcm -1) (10). Reactions of L-tryptophan were measured using the coupled assay with lactate dehydrogenase and NADH, measured at 340 n m ( e = 6.22 x 103 M - l c m -1) (11). Concentrations of wild-type and W248F were determined from A 1% = 8.9 for wild-type and A 1% = 8.0 for W248F at 278 nm, assuming a subunit molecular weight of 52,242 (2). The absorption coefficients of wild-type and W248F were determined from amino acid analysis. CD and fluorescence spectra were measured with a Jasco J600 spectropolarimeter and a Hitachi F-4010 fluorescence spectrophotometer, respectively. Enzyme assays were performed using a Hitachi 220S spectrophotometer at 30°C. The temperature was controlled with a thermostatically controlled cell holder.
Instrumentation.
RESULTS
AND
DISCUSSION
W248F was found to be overproduced from E. coli K-12 MD55 cells harboring the mutant plasmid pMD6BW248F, as was the wild type tryptophanase (8). About 200 mg of W248F was purified from 1 L of medium. In the present study, the wild and mutant enzymes were further purified by using an affinity column, SP-C-Ala. The specific activities of wild-type and W248F thus purified were 55 and 46 units/mg, respectively, for SOPC at pH 7.8 and 30°C. Figure 1 shows CD spectra of wild-type and W248F enzymes. It was found that the secondary structure is the same in both enzymes. At around 370 nm, however, ellipticity of W248F was smaller than that of wild-type, indicating that the micro-environment of PLP in the mutant differs slightly from that of the wild enzyme. Figure 2a shows tryptophyl fluorescence spectra of wild-type and W248F. Fluorescence energy transfer from Trp to PLP at around 500 nm was not observed for W248F. Tokushige et al. (12) also reported the same results using chemical modification. Figure 2b shows excitation-emission maps of holo
100
15 o E
10 50
E ¢N
o x ~:~
-10
2"
-15 2OO
250
~
-5o
, 300
350
~ 400
-100 450
500
Wavelength (nm)
Fig. 1. CD spectra of wild-type (solid line) and W248F (broken line) tryptophanases at 37°C. The buffer used was 50 mM potassium phosphate, pH 7.8, containing 60 btM PLP, 1 mM dithiothreitol, and 1 mM EDTA. 758
Vol.
1 7 3 , N o . 2, 1 9 9 0
B I O C H E M I C A L A N D B I O P H Y S I C A L RESEARCH C O M M U N I C A T I O N S
200
20
150
15
o -= IL 100
10
a
._>= 50 r¢ 0 300
i 350
i 400
Wavelength
b
",. . . . .
I
450
500
550
: 0
600
(nm)
400 .....
" ' r
".o;o-W2'sF:
E" ¢- 350
"~ 300 (J x uJ
o
250
400
E r-
350
,~
300
25O
300
350
400
Emission
450
500
550
(nm)
300
350
400
Emission
450
500
550
(rim)
Fig. 2. Tryptophyl fluorescence spectra (a) and excitation-emission maps (b) of wild-type and W248F tryptophanases at 25°C. Protein concentration was 50 gg/mL. (a): Excitation wavelength was 295 nm. Solid line, wild-type; broken line, W248F. (b): Fluorescence spectra were measured at 5-nm intervals of excitation wavelength and represented in contour. The two cross lines at the upper left-hand and lower right-hand comers in maps were due to primary and secondary scattering, respectively. The buffer used was 100 mM potassium phosphate, pH 7.8. Holo enzymes were measured immediately after removing excess PLP with a Sephadex G-25 column. Apo enzymes were prepared using DL-penicillamine according to the method of Morino and Snell (19).
and apo enzymes of wild-type and W248F. The difference in the fluorescence energy transfer regions (excitation; at around 260 n m - emission; at around 500 nm) between the maps of holo wild-type and holo W248F is clearly shown. The fluorescence due to PLP (excitation; at around 335 n m - emission; at around 380 nm) disappeared in the maps of apo enzymes. These fluorescence maps also suggest that global structures of wild-type and W248F in holo and apo forms are quite indistinguishable. 759
Vol. 173, No. 2, 1990
BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS
7O
E
6O
=
50
i
i
i
4O
"f ao u x-
~
2O
0
I
7
,
I
I
8
9
pH Fig. 3. Activity-pH profiles of wild-type and W248F tryptophanases. Specific activities of wild-type (solid circle) and W248F (open circle) were measured for 0.33 mM SOPC in 0.1 M potassium phosphate buffer containing 60 ktM of PLP at various pH. The amount of enzyme used in an assay was 10 ~tg.
In order to clarify the effect of the replacement toward PLP-binding, dissociation constants (Kd) for PLP were determined (13). The Kd value of W248F was increased tenfold compared with that of wild-type; 2.5 I.tM for W248F and 0.25 gM for wild-type. Ozone-oxidation of Trp 248 to N'-formyl kynurenine also decreased the affinity towards PLP (4). These observations indicate that Trp 248 in the active site interacts closely with PLP. In the presence of excess PLP, specific activities of wild-type and W248F for SOPC were measured at various pH as shown in Fig. 3. The specific activity of W248F at optimum pH 8.0 was approximately 85% of wild-type. In order to study the enzymatic reaction in detail, further kinetic analyses were performed. As shown in Table 1, although the Km values for SOPC and L-tryptophan were identical in both enzymes, the kcat values of the mutant were decreased to 85% of that of the wild-type. These findings indicate that Trp 248 plays an important role in the transition state of the enzymatic reaction but not in the ground state (binding state of substrates).
Table 1. Kinetic parameters for wild-type and W248F tryptophanases Enzyme
SOPC
L-Trp
kc~ (S1)
Km (mM)
kc~/Km (M'ls -1)
Wild-type
58
0.043
W248F
51 0.88
W248F/Wild
PLP
k,:~ (s"1)
Krn (mM)
/¢~/Km (M'ls -1 )
Kd (~LM)
1.34x10s
5.0
0.12
4.19xl 0 4
0.25
0.044
1.16x106
4.3
0.13
3.28x104
2.5
1.0
0.86
0.86
1.1
0.78
10
760
VOI. 173, No. 2, 1990
BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS
Many PLP-dependent enzymes have a Trp in the active site and similar mutagenic experiments have been performed. In aspartate aminotransferase (14) and D-amino acid transaminase (15), replacement of the tryptophan residue resulted in changes in kcat and/or Km in the enzymatic reaction. In the X-ray crystallographic structure of aspartate aminotransferase (16, 17, 18), it was found that the indole ring of Trp 140 in the active site interacts with the pyridine ring of PLP and that slight conformation changes of the two rings were observed upon binding of a pseudo-substrate. In the case of tryptophanase, since Trp 248 was shown to interact closely with PLP, it is suggested that the Trp 248 also plays an important role in determining the correct orientation of a substrate in the transition state of the catalytic mechanism. Because no changes in Km value occurred upon replacement, the indole NH proton of Trp 248 is probably not involved in hydrogen bonding to the substrate in contrast to the case of aspartate aminotransferase. ACKNOWLEDGMENT
We wish to thank Professor Kenji Soda of the Institute for ChemicalResearch, Kyoto Universityfor kindlyallowingus to performCD measurements.
REFERENCES
(1) Snell, E. E. (1975) Adv. Enzymol. 42, 287-333. (2) Tokushige, M., Tsujimoto, N., Oda, T., Honda, T., Yumoto, N., Ito, S., Yamamoto, M., Kim, E. H., and Hiragi, Y. (1989) Biochimie 71, 711-720. (3) Deely, M. C., and Yanofsky, C. (1981) J. BacterioL 147, 787-796. (4) Tokushige, M., Fukada, Y., and Watanabe, Y. (1979) Biochem. Biophys. Res. Commun. 86, 976-981. (5) Phillips, R. S., Ravichandran, K., and Tersch, R. L. (1989) Enzyme Microb. TechnoL, 11, 80-83. (6) Taylor, J. W., Ott, J., and Eckstein, F. (1985) Nucleic Acids Res. 13, 8765-8785. (7) Sanger, F., Nicklen, S., and Coulson, A. R. (1977) Proc. Natl. Acad. Sci. U. S. A. 80, 3963-3965. (8) Tani, S., Tsujimoto, N., Kawata, Y., and Tokushige, M. (1990) Biotechnol. AppL Biochem. 12, 28-33. (9) Ikeda, S., Hara, H., and Fukui, S. (1974) Biochim. Biophys. Acta, 372, 400-406. (10) Suelter, C. H., Wang, J., and Snell, E. E. (1976) FEBS Lett. 66, 230-232. (11) Morino, Y., and Snell, E. E. (1970) Methods Enzymol. 17A, 439-446. (12) Tokushige, M., Iimuma, K., Yamamoto, M., and Nishijima, Y.(1980) Biochem. Biophys. Res. Commun. 96, 863-869. (13) Kazarinoff, M. N. and Snell, E. E. (1977) J. BioL Chem. 252, 7598-7602. (14) Hayashi, H., Inoue, Y., Kuramitsu, S., Morino, Y., and Kagamiyama, H. (1990) Biochem. Biophys. Res. Commun. 167, 407-412. 761
Vol. 173, No. 2, 1990
BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS
(15) Pozo, A. M., Merola, M., Ueno, H., Manning, J. M., Tanizawa, K., Nishimura, K., Asano, S., Tanaka, H., Soda, K., Ringe, D., and Petsko, G. A. (1989)Biochemistry 28, 510-516. (16) Amone, A., Rogers, P. H., Hyde, C. C., Briley, P. D., Metzler, C. M., and Metzler, D. E. (1985) in : Transaminases (Christen, P., and Metzler, D. E. eds) pp. 138-155, Wiley, New York. (17) Jansonius, J. N., Eichele, G., Ford, G. C., Picot, D., Thaller, C., and Vincent, M. G. (1985) in : Transaminases (Christen, P., and Metzler, D. E. eds) pp. 110-138, Wiley, New York. (18) Kamitori, S., Okamoto, A., Hirotsu, K., Higuchi, T., Kuramitsu, S., Kagamiyama, H., Matsuura, Y., and Katsube, Y. (1990) J. Biochem. 108, 175-184. (19) Morino, Y., and Snell, E. E. (1967) J. Biol. Chem. 242, 5591-5601.
762
BIOCHEMICAL AND BIOPHYSICALRESEARCH COMMUNICATIONS Pages 756-762
December 14, 1990
ROLE OF TRYPTOPHAN 248 OF TRYPTOPHANASE FROM
IN THE ACTIVE SITE ESCHERICHIA COLI 1
Yasushi Kawata 2, Nobuharu Tsujimoto, Shunsuke Tani, Tomohiro Mizobata, and Masanobu Tokushige 3 Department of Chemistry, Faculty of Science, Kyoto University, Kyoto 606, Japan Received November 8, 1990
Tryptophan 248, located in the active site of tryptophanase from Escherichia coli, has been replaced with phenylalanine by site-directed mutagenesis. Judging from CD and fluorescence spectra, the global structure of the mutant enzyme was found to be the same as that of the wild-type enzyme. The binding affinity of the mutant enzyme for the coenzyme pyridoxal Y-phosphate (PLP) was reduced tenfold compared to the wild-type enzyme. Kinetic analyses under PLP-saturated conditions indicated that the Km values of the mutant enzyme for substrates are the same as those of wild-type enzyme but the kcat values are decreased to about 85%, which accounts for the overall activity decrease. These findings suggest that tryptophan 248 interacts closely with PLP and plays an important role in the catalytic reaction. ~ 199oAoademioP..... ~no.
Tryptophanase (L-tryptophan indole-lyase, EC 4.1.99.1) catalyses the reversible degradation of tryptophan to indole, pyruvate, and ammonia (1). The enzyme from E. coli consists of four identical subunits with a molecular weight of 52,242 (total molecular weight 208,968), as determined by DNA sequence analysis (2, 3). Each subunit binds one pyridoxal 5'-phosphate (PLP) molecule
1This work was supported in part by Grants-in-Aid for Scientific Research (No.01780198) from the Ministry of Education, Science and Culture of Japan and also by Amano Pharmaceutical Co. Foundation. 2To whom correspondence should be addressed. 3Deceased October 4, 1990. Abbreviations: CD, circular dichroism; PLP, pyridoxal 5'-phosphate; SOPC, S-o-nitrophenyl-L-cysteine; W248F, mutant tryptophanase in which tryptophan 248 has been replaced with phenylalanine. 0006-291X/90 $1.50 Copyright © 1990 by Academic Press, Inc. All rights of reproduction in any form reserved.
756
Vol. 173, No. 2, 1990
BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS
to its active site as a coenzyme. Tryptophanase possesses two tryptophans in its amino acid sequence. One of these, Trp 248, has been previously reported to be located in the active site region (4). In order to study the role of this tryptophan residue in the catalytic mechanism, we have prepared a mutant enzyme by sitedirected mutagenesis, in which Trp 248 was replaced with phenylalanine (W248F). From the measurements of PLP-binding affinity and kinetic analyses of enzymatic action, it was indicated that Trp 248 interacts closely with the PLP and plays an important role in the catalytic reaction. MATERIALS AND METHODS
Chemicals. L-tryptophan, NADH, PLP, and lactate dehydrogenase from hog muscle were purchased from nacalai tesque. S-o-nitrophenyl-L-cysteine (SOPC) was prepared by the reaction of L-cysteine hydrochloride hydrate with 2fluoronitrobenzene in dimethylformamide containing triethylamine (5). Aminohexyl-Sepharose was obtained from Pharmacia (Uppsala, Sweden). All other chemicals were of the highest grade commercially available. Preparation of W248F mutant. A 220-base pair PstI/KpnI fragment was subcloned from pMD6B which encodes the E. coli B/lt7-A tryptophanase (2) into M13mpl9, and single-stranded DNA was prepared. Oligonucleotide directed sitespecific mutagenesis was performed by the method of Eckstein (6) using a kit from Amersham Corp. The mutagenic oligonucleotide (5'-CTC GAT GGT GAA GTC TTT GTA-3') was synthesized using an Applied Biosystems DNA synthesizer (model 381A). Candidate plaques were screened for the mutation by digestion of the plasmid with AvalI (the mutation lacks the restriction site), and the mutated DNA fragment was sequenced by the dideoxy method (7). Doublestranded RF DNA was prepared from the mutant phage and the 220-base pair PstI/KpnI fragment was isolated and ligated at the original PstI/KpnI site of pMD6B. To insure that the desired mutation had been introduced, the resulting plasmid (pMD6BW248F) was sequenced again in the vicinity of the mutation and the ligation sites by subcloning of a 627-base pair HhaI fragment to M13mp19. The pMD6BW248F was then transformed into E. coli K-12 MD55, which is an auxotroph of tryptophan. The cells were grown and the enzyme was purified as described previously (8). In the present experiments, wild-type and W248F were further purified by using an affinity column of Sepharose-bound PLP-alanine complex, SP-C-Ala. SP-C-Ala was prepared by the method of Ikeda et al. (9) with modifications. The enzyme (20 mg) was applied to the column (0.5 x 10 cm) previously equilibrated with 10 mM potassium phosphate buffer, pH 7.0 and washed thoroughly with 1 M potassium phosphate buffer, pH 7.0, and then eluted with 50 mM potassium acetate, pH 5.0, containing 50 ktM PLP. The buffer of the eluted enzyme was immediately substituted with 20 mM potassium phosphate buffer, pH 7.0, containing 0.5 mM PLP and 5 mM dithiothreitol. The specific activity of the enzyme obtained thus was improved. Enzyme assays. Routine enzyme assays were performed with 0.33 mM SOPC in 0.1 M potassium phosphate buffer, pH 7.8, at 30°C, measured at 370 n m ( e = 757
Vol. 173, No. 2, 1990
BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS
1.86 x 103 M-lcm -1) (10). Reactions of L-tryptophan were measured using the coupled assay with lactate dehydrogenase and NADH, measured at 340 n m ( e = 6.22 x 103 M - l c m -1) (11). Concentrations of wild-type and W248F were determined from A 1% = 8.9 for wild-type and A 1% = 8.0 for W248F at 278 nm, assuming a subunit molecular weight of 52,242 (2). The absorption coefficients of wild-type and W248F were determined from amino acid analysis. CD and fluorescence spectra were measured with a Jasco J600 spectropolarimeter and a Hitachi F-4010 fluorescence spectrophotometer, respectively. Enzyme assays were performed using a Hitachi 220S spectrophotometer at 30°C. The temperature was controlled with a thermostatically controlled cell holder.
Instrumentation.
RESULTS
AND
DISCUSSION
W248F was found to be overproduced from E. coli K-12 MD55 cells harboring the mutant plasmid pMD6BW248F, as was the wild type tryptophanase (8). About 200 mg of W248F was purified from 1 L of medium. In the present study, the wild and mutant enzymes were further purified by using an affinity column, SP-C-Ala. The specific activities of wild-type and W248F thus purified were 55 and 46 units/mg, respectively, for SOPC at pH 7.8 and 30°C. Figure 1 shows CD spectra of wild-type and W248F enzymes. It was found that the secondary structure is the same in both enzymes. At around 370 nm, however, ellipticity of W248F was smaller than that of wild-type, indicating that the micro-environment of PLP in the mutant differs slightly from that of the wild enzyme. Figure 2a shows tryptophyl fluorescence spectra of wild-type and W248F. Fluorescence energy transfer from Trp to PLP at around 500 nm was not observed for W248F. Tokushige et al. (12) also reported the same results using chemical modification. Figure 2b shows excitation-emission maps of holo
100
15 o E
10 50
E ¢N
o x ~:~
-10
2"
-15 2OO
250
~
-5o
, 300
350
~ 400
-100 450
500
Wavelength (nm)
Fig. 1. CD spectra of wild-type (solid line) and W248F (broken line) tryptophanases at 37°C. The buffer used was 50 mM potassium phosphate, pH 7.8, containing 60 btM PLP, 1 mM dithiothreitol, and 1 mM EDTA. 758
Vol.
1 7 3 , N o . 2, 1 9 9 0
B I O C H E M I C A L A N D B I O P H Y S I C A L RESEARCH C O M M U N I C A T I O N S
200
20
150
15
o -= IL 100
10
a
._>= 50 r¢ 0 300
i 350
i 400
Wavelength
b
",. . . . .
I
450
500
550
: 0
600
(nm)
400 .....
" ' r
".o;o-W2'sF:
E" ¢- 350
"~ 300 (J x uJ
o
250
400
E r-
350
,~
300
25O
300
350
400
Emission
450
500
550
(nm)
300
350
400
Emission
450
500
550
(rim)
Fig. 2. Tryptophyl fluorescence spectra (a) and excitation-emission maps (b) of wild-type and W248F tryptophanases at 25°C. Protein concentration was 50 gg/mL. (a): Excitation wavelength was 295 nm. Solid line, wild-type; broken line, W248F. (b): Fluorescence spectra were measured at 5-nm intervals of excitation wavelength and represented in contour. The two cross lines at the upper left-hand and lower right-hand comers in maps were due to primary and secondary scattering, respectively. The buffer used was 100 mM potassium phosphate, pH 7.8. Holo enzymes were measured immediately after removing excess PLP with a Sephadex G-25 column. Apo enzymes were prepared using DL-penicillamine according to the method of Morino and Snell (19).
and apo enzymes of wild-type and W248F. The difference in the fluorescence energy transfer regions (excitation; at around 260 n m - emission; at around 500 nm) between the maps of holo wild-type and holo W248F is clearly shown. The fluorescence due to PLP (excitation; at around 335 n m - emission; at around 380 nm) disappeared in the maps of apo enzymes. These fluorescence maps also suggest that global structures of wild-type and W248F in holo and apo forms are quite indistinguishable. 759
Vol. 173, No. 2, 1990
BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS
7O
E
6O
=
50
i
i
i
4O
"f ao u x-
~
2O
0
I
7
,
I
I
8
9
pH Fig. 3. Activity-pH profiles of wild-type and W248F tryptophanases. Specific activities of wild-type (solid circle) and W248F (open circle) were measured for 0.33 mM SOPC in 0.1 M potassium phosphate buffer containing 60 ktM of PLP at various pH. The amount of enzyme used in an assay was 10 ~tg.
In order to clarify the effect of the replacement toward PLP-binding, dissociation constants (Kd) for PLP were determined (13). The Kd value of W248F was increased tenfold compared with that of wild-type; 2.5 I.tM for W248F and 0.25 gM for wild-type. Ozone-oxidation of Trp 248 to N'-formyl kynurenine also decreased the affinity towards PLP (4). These observations indicate that Trp 248 in the active site interacts closely with PLP. In the presence of excess PLP, specific activities of wild-type and W248F for SOPC were measured at various pH as shown in Fig. 3. The specific activity of W248F at optimum pH 8.0 was approximately 85% of wild-type. In order to study the enzymatic reaction in detail, further kinetic analyses were performed. As shown in Table 1, although the Km values for SOPC and L-tryptophan were identical in both enzymes, the kcat values of the mutant were decreased to 85% of that of the wild-type. These findings indicate that Trp 248 plays an important role in the transition state of the enzymatic reaction but not in the ground state (binding state of substrates).
Table 1. Kinetic parameters for wild-type and W248F tryptophanases Enzyme
SOPC
L-Trp
kc~ (S1)
Km (mM)
kc~/Km (M'ls -1)
Wild-type
58
0.043
W248F
51 0.88
W248F/Wild
PLP
k,:~ (s"1)
Krn (mM)
/¢~/Km (M'ls -1 )
Kd (~LM)
1.34x10s
5.0
0.12
4.19xl 0 4
0.25
0.044
1.16x106
4.3
0.13
3.28x104
2.5
1.0
0.86
0.86
1.1
0.78
10
760
VOI. 173, No. 2, 1990
BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS
Many PLP-dependent enzymes have a Trp in the active site and similar mutagenic experiments have been performed. In aspartate aminotransferase (14) and D-amino acid transaminase (15), replacement of the tryptophan residue resulted in changes in kcat and/or Km in the enzymatic reaction. In the X-ray crystallographic structure of aspartate aminotransferase (16, 17, 18), it was found that the indole ring of Trp 140 in the active site interacts with the pyridine ring of PLP and that slight conformation changes of the two rings were observed upon binding of a pseudo-substrate. In the case of tryptophanase, since Trp 248 was shown to interact closely with PLP, it is suggested that the Trp 248 also plays an important role in determining the correct orientation of a substrate in the transition state of the catalytic mechanism. Because no changes in Km value occurred upon replacement, the indole NH proton of Trp 248 is probably not involved in hydrogen bonding to the substrate in contrast to the case of aspartate aminotransferase. ACKNOWLEDGMENT
We wish to thank Professor Kenji Soda of the Institute for ChemicalResearch, Kyoto Universityfor kindlyallowingus to performCD measurements.
REFERENCES
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