J. Am. Chem. Soc. 1998, 120, 451-452
451
the pH dependence of the mutants are relatively unaffected by
fluorine substitution with the exception of the 2,3,5,6-tetrafluo-
rotyrosine mutant. The results are consistent with a previously
proposed hydrogen-bonding role for Tyr9 in human GST
A1-14,7 and are compared with previous experimental8 and
theoretical studies9 of GSTs in which the pKa of the active tyrosine
residue is altered.
Analysis of the Role of the Active Site Tyrosine in
Human Glutathione Transferase A1-1 by Unnatural
Amino Acid Mutagenesis
Jon S. Thorson,† Injae Shin, Eli Chapman, Gun Stenberg,‡
Bengt Mannervik,*,‡ and Peter G. Schultz*
A series of fluorinated tyrosine analogues, 2-fluorotyrosine (1),
3-fluorotyrosine (2), 3,5-difluorotyrosine (3), and 2,3,5,6-tet-
rafluorotyrosine (4), was substituted for Tyr9.10,11 Due to
fluorine’s relatively small size (van der Waals radius of 1.35 Å
relative to 1.2 Å for hydrogen),12 the steric perturbation resulting
from fluorine substitution should be relatively small. Incorpora-
tion of the fluorinated tyrosine analogues 1-4, as well as
L-tyrosine (5), at position 9 of human GST A1-1 was ac-
complished by in vitro suppression of a Tyr9 f TAG stop
mutation with a chemically aminoacylated suppressor tRNACUA
Department of Biochemistry, Uppsala UniVersity
Biomedical Center, Box 576, S-751 23 Uppsala, Sweden
Department of Chemistry
Howard Hughes Medical Institute
UniVersity of California, Berkeley, California 94720
ReceiVed September 10, 1997
A major detoxification pathway used by aerobic organisms
involves the enzymatic conjugation of the tripeptide glutathione
(GSH) to the electrophilic center of toxic substances by the
glutathione transferases (GSTs) (EC 2.5.1.18).1 These enzymes
activate the cysteine thiol group of GSH for nucleophilic addition
to a variety of substrates, including aryl halides, R,â-unsaturated
aldehydes and ketones, and epoxides. However, despite a large
number of biochemical and structural studies,2 the mechanism
by which GSH transferases catalyze these addition reactions
remains unclear. The three-dimensional structures have shown
that the hydroxyl group of an active site tyrosine residue (Tyr9
for human GST A1-1, class alpha), which is conserved among
the majority of known GSH transferases, is within hydrogen-
bonding distance of the sulfur of glutathione.2 In solution, the
pKa of GSH is about 9.0,3 whereas in the GST A1-1 enzyme-
GSH complex the pKa of the thiol group is 6.2.4 The tyrosine
residue of this complex is believed to stabilize the thiolate of
GSH through a hydrogen-bonding interaction (TyrOH‚‚‚-SG).5
Alternatively, the abnormally low pKa of the tyrosyl hydroxyl
group may provide a tyrosinate anion which can act as a general
base (TyrO-‚‚‚HSG)6 to abstract the proton from the sulfhydryl
group. To investigate this issue, we have used unnatural amino
acid mutagenesis to site-specifically replace Tyr9 in human GST
A1-1 with a series of fluorinated tyrosine analogues with pKa
values ranging from 5.3 to 10. The observed values of kcat and
derived from yeast tRNAPhe 13,14
Amino acids 1-4 were incor-
.
porated with suppression efficiencies ranging from 15 to 25%
relative to that for in vitro expression of wild-type (wt) protein.
In contrast, when tRNACUA was omitted from the in vitro reaction
or was not aminoacylated, less than 1% full-length GST was
produced in comparison to in vitro expression of wt GST. Wild-
type and mutant GSTs were purified to homogeneity from 5.0
mL in vitro protein synthesis reactions.15
Steady-state kinetic analysis of the wt and mutant enzymes
was performed over a pH range of 6.0-8.0 using previously
described assay conditions.16 Interestingly, the increased acidity
of the active site hydroxyl group significantly influences the
catalytic properties only of the Tyr9 f 2,3,5,6-tetrafluorotyrosine
mutant (Figure 1). The limiting value of kcat/KmCDNB on the high-
pH plateau, which is postulated to reflect the reactivity of the
monoprotonated species of the complex,8 declines only an average
of approximately 2-fold for the Tyr9 f 1, Tyr9 f 2, and Tyr9
f 3 mutants. In contrast, the kcat/KmCDNB of mutant 4 is reduced
roughly 20-fold in the same pH range. The pH dependence of
log kcat/Km for the mutants is also very similar to that of wt
enzyme, again with the exception of 2,3,5,6-tetrafluorotyrosine,
(7) Widersten, M.; Bjo¨rnestedt, R.; Mannervik, B. Biochemistry 1996, 35,
7731-7742.
† Current address: Laboratory for Biosynthetic Chemistry, Memorial Sloan-
Kettering Institute for Cancer Research, 1275 York Avenue, New York, New
York 10021.
(8) Parsons, J. F.; Armstrong, R. N. J. Am. Chem. Soc. 1996, 118, 2295-
2296.
(9) Zheng, Y.-J.; Ornstein, R. L. J. Am. Chem. Soc. 1997, 119, 1523-
1528.
‡ Uppsala University.
(1) (a) Armstrong, R. N. Crit. ReV. Biochem. 1987, 22, 39-88. (b)
Mannervik, B.; Danielson, U. H. Crit. ReV. Biochem. 1988, 23, 283-337. (c)
Armstrong, R. N. Chem. Res. Toxicol. 1991, 4, 131. (d) Armstrong, R. N.
AdV. Enzymol. Relat. Areas Mol. Biol. 1994, 69, 1-44. (e) Mannervik, B.;
Widersten, M. In AdVances in Drug Metabolism in Man (Pacifici, G. M.,
Fracchia, G. N., Eds.; European Commission: Luxembourg, 1995; pp 407-
459.
(2) (a) Ji, X.; Zhang, P.; Armstrong, R. N.; Gilliland, G. L. Biochemistry
1992, 31, 10169-10184. (b) Xiao, G.; Liu, S.; Ji, X.; Johnson, W. W.; Chen,
J.; Parsons, J. F.; Stevens, W. J.; Gilliland, G. L.; Armstrong, R. N.
Biochemistry 1996, 35, 4753-4765. (c) Ji, X.; Armstrong, R. N.; Gilliland,
G. L. Biochemistry 1993, 32, 12949-12954. (d) Sinning, I.; Gilliland, G. L.;
Armstrong, R. N.; Ji, X.; Board, P. G.; Olin, B.; Mannervik, B.; Jones, T. A.
J. Mol. Biol. 1993, 232, 192-212. (e) Ji, X.; von Rosenvinge, E. C.; Johnson,
W. W.; Tomarey, S. I.; Piatigorsky, J.; Armstrong, R. N.; Gilliland, G. L.
Biochemistry 1995, 34, 5317-5328. (f) Ji, X.; von Rosenvinge, E. C.; Johnson,
W. W.; Armstrong, R. N.; Gilliland, G. L. Proc. Natl. Acad. Sci. U.S.A. 1996,
93, 8208-8213.
(3) (a) Novak, M.; Lin, J. J. Am. Chem. Soc. 1996, 118, 1302-1308. (b)
Cheesman, B. V.; Arnold, A. P.; Rabenstein, D. L. J. Am. Chem. Soc. 1988,
110, 6359-6364.
(4) Bjo¨rnestedt, R.; Stenberg, G.; Widersten, M.; Board, P. G.; Sinning,
I.; Jones, T. A.; Mannervik, B. J. Mol. Biol. 1995, 247, 765-773.
(5) Liu, S.; Zhang, P.; Ji, X.; Johnson, W. W.; Gilliland, G. L.; Armstrong,
R. N. J. Biol. Chem. 1992, 267, 4296-4299.
(6) (a) Atkins, W. M.; Wang, R. W.; Bird, A. W.; Newton, D. J.; Lu, A.
Y. H. J. Biol. Chem. 1993, 268, 19188-19191. (b) Karshikoff, A.; Reinemer,
P.; Huber, R.; Ladenstein, R. Eur. J. Biochem. 1993, 125, 663-670. (c) Meyer,
D. J.; Xia, C.; Coles, B.; Chen, H.; Reinemer, P.; Huber, R.; Ketterer, B.
Biochem. J. 1993, 293, 351-356.
(10) Amino acids 1, 2, 4, and 5 are commercially available. 3,5-
Difluorotyrosine was synthesized from 3,5-difluoro-4-methoxybenzaldehyde
by the procedure of Kruse et al.20 The aldehyde was reduced with sodium
borohydride followed by bromination of the hydroxyl group with PBr3. The
bromide was subsequently coupled with diethyl acetamidomalonate, and the
resulting product was hydrolyzed with 50% HBr to give the desired product:
1H NMR (D2O) δ 6.70 (2H, d, J ) 8.0 Hz), 3.72 (1H, dd, J ) 7.4, 5.4 Hz),
2.96 (1H, dd, J ) 14.7, 5.2 Hz), 2.83 (1H, dd, J ) 14.7, 7.8 Hz); HR MS
calcd 218.0629, found 218.0626 (M + 1). It has been shown that only L-amino
acids are accepted by the amino acid biosynthetic machinery.14
(11) Handbook of Biochemistry and Molecular Biology: Physical and
Chemical Data; Fasman, G. D., Ed.; CRC Press: Boca Raton, FL, 1976; Vol
I, p 314.
(12) Handbook of Chemistry and Physics; West, R. C., Ed.; CRC Press:
Cleveland, OH, 1976; p D178.
(13) Plasmid pAED4-GST.TAG9 was derived from plasmid pAED4-
GST.WT which contains the GST A1-1 gene under T7 control. Plasmid
pAED4-GST.WT was constructed by PCR gene amplification from plasmid
pKHA116b and ligation into plasmid pAED4 (New England Biolabs, Waltham,
MA).
(14) Thorson, J. S.; Cornish, V. W.; Barrett, J. E.; Cload, S. T.; Yano, Y.;
Schultz, P. G. Methods in Molecular Biology; Humana Press: U.K. In press.
(15) Wild-type and mutant enzymes were purified by affinity chromato-
graphy5,16b in 50 mM Tris‚HCl (pH 7.4), 200 mM NaCl, 0.5 mM DTT followed
by elution with the same buffer containing 50 mM GSH. The enzyme was
stored and assayed in 100 mM sodium phosphate (pH 6.5) containing 1 mM
EDTA.
(16) (a) Habig, W. H.; Jakoby, W. B. Methods Enzymol. 1981, 77, 398-
405. (b) Stenberg, G.; Bjo¨rnestedt, R.; Mannervik, B. Protein Express. Purif.
1992, 3, 80-84.
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