Semisynthetic tellurosubtilisin with glutathione peroxidase activity

Article abstract of DOI:10.1021/ja052451v

Reaction of the hydroxyl group of serine-221 of subtilisin with phenylmethanesulfonylfluoride followed by nucleophilic substitution with sodium hydrogen telluride, a semisynthetic telluroprotein, tellurosubtilisin, was prepared. Tellurosubtilisin, which displays high substrate specificity for aromatic thiols, exhibits remarkable peroxidase activity and catalyzes the reduction of hydrogen peroxide by 3-carboxy-4-nitrobenzenethiol 20000 times more efficiently than diphenyl diselenide. Copyright

Full text of DOI:10.1021/ja052451v

Published on Web 07/29/2005
Semisynthetic Tellurosubtilisin with Glutathione Peroxidase Activity
Shizhong Mao, Zeyuan Dong, Junqiu Liu,* Xiangqiu Li, Xiaoman Liu, Guimin Luo, and Jiacong Shen
Key Laboratory for Supramolecular Structure and Materials of Ministry of Education, Jilin UniVersity,
Changchun 130012, People’s Republic of China
Received April 15, 2005; E-mail: junqiuliu@jlu.edu.cn
There has been increasing interest in mimicking the functions
of an antioxidative selenoenzyme, glutathione peroxidase (GPx, EC
1.11.1.9), which catalyzes the reduction of a variety of hydroper-
oxides (ROOH) using glutathione (GSH) as the reducing substrate
(Scheme 1), and thus protects cells from oxidative damage.1 Owing
to its biological importance, considerable efforts have been devoted
to produce organoselenium compounds which mimic the properties
of GPx in recent years.2
Scheme 1. Catalytic Cycle for GPx
Figure 1. (A) UV spectra of isolated Enz-TeSAr in 50 mM PIPES buffer
containing 10 mM CaCl2, pH 7.0 (ꢀ326 ) 7600 M-1 cm-1). (B) After
addition of excess DTT (for ArSH, pH 7.0, ꢀ410 ) 13 600 M-1 cm-1).
phenylmethanesulfonylfluoride (PMSF, 150 µL of a 20 mg mL-1
solution in acetonitrile) for 1 h at 25 °C. The sulfonylated enzyme
solution was treated with 0.5 mL of 1 M sodium hydrogen telluride
(NaHTe) solution, prepared according to the procedure of Akiba
et al.10 in a well-ventilated hood for safety, for 36 h at 40 °C under
nitrogen. The resulting telluroprotein was aerially oxidized and then
Besides molecular biology techniques for gene manipulation and
functional selection toward enzyme design, an alternative strategy
purified by gel filtration on Sephadex G-25 and affinity chroma-
for generating an efficient enzyme model is chemical incorporation
tography on thiopropyl-Sepharose 6B. The generated protein was
of catalytic groups into naturally existing or artificially generated
homogeneous, as judged by polyacrylamide gel electrophoresis,11
substrate binding scaffolds.3 This strategy has been demonstrated
and the yield of telluroprotein was typically 10%, based on the
very efficiently in the construction of GPx models by chemically
amounts of the starting protein. As a reference experiment, subtilisin
introducing catalytic selenocysteine into thiol glutathione-specific
was directly treated by NaHTe without initial PMSF activation,
antibody and bioimprinted protein by our group.4 Natural proteins
and no detectable protein was observed on thiol-mediated affinity
also provide versatile templates for chemical manipulation. Fol-
chromatography, indicating that the tellurium substitution took place
lowing the first semisynthetic thiol subtilisin,5 Hilvert and co-
at active serine-221 of subtilisin, similar to that previously reported
workers developed a methodology to introduce selenium into the
binding pocket of subtilisin to yield selenosubtilisin.6 This hybrid
protein combines the intrinsic chemistry of selenium to show GPx
activity with the binding specificity of subtilisin for aromatic groups.
for thiol/selenosubtilisin.5,6
The molecular weight (Mr) value was found to be 27 432 ( 5
by MALDI-TOF-MS, which was consistent with that of the
expected tellurinic acid form of the enzyme (Enz-TeO2H, calculated
Recent studies showed that organotellurium compounds also exhibit
Mr ) 27 432). The oxidized enzyme reacted with 3 equiv of
3-carboxy-4-nitrobenzenethiol (ArSH) to produce the tellurenyl
sulfide intermediate (Enz-TeSAr) that can be reduced by dithio-
threitol (DTT) to release an equivalent of ArSH (Figure 1), in
analogy to the reduction of tellurocyclodextrin by ArSH,7f also
indicating that it exists as the tellurinic acid form. About 1 equiv
(0.97 ( 0.4) of tellurium per molar of subtilisin was incorporated,
as judged by anaerobic titration of the reduced enzyme with 5,5′-
dithiobis(2-nitrobenzoic acid).12 One might expect that the tellurium
substitution of the hydroxyl group of serine-221 should have no
effect on the structure of subtilisin, and the identical behaviors of
circular dichroism (CD) spectra for subtilisin and tellurosubtilisin
verified this speculation.11
Like natural GPx, tellurosubtilisin can catalyze the reduction of
ROOH by thiols. The catalytic activity was assessed in detail
according to a modified method reported by Hilvert et al.6 using
ArSH as the GSH alternative. The initial rates for the reduction of
H2O2 by ArSH in 100 mM 4-morpholineethanesulfonic acid (MES)
buffer (pH 5.5) in the presence of tellurosubtilisin were determined
at 25 °C by monitoring the disappearance of ArSH at 410 nm. To
similar catalytic actions.7 To our knowledge, however, tellurium
has not been found in wild-type proteins, although tellurium had
been bioincorporated to protein for structure analysis.8 Herein, we
reported the preparation of the first semisynthetic telluroenzyme,
tellurosubtilisin, which was generated by chemically introducing
tellurium into the binding site of subtilisin and then acted as an
excellent GPx mimic.
Subtilisin is a bacterial serine protease [EC 3.4.21.14] that is
chemically and structurally well-studied.9 Along with the successful
chemical modification of subtilisin to thiol/selenosubtilisin,5,6 the
semisynthetic GPx mimic, tellurosubtilisin, was prepared via a three-
step protocol starting from subtilisin Carlsberg (Scheme 2). The
hydroxyl group of serine-221 of subtilisin (150 mg in 10 mL of 50
mM 1,4-piperazinediethanesulfonic acid (PIPES) buffer containing
10 mM CaCl2, pH 7.0) was specifically activated by reaction with
Scheme 2. Chemical Conversion of Subtilisin into Tellurosubtilisin
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10.1021/ja052451v CCC: $30.25 © 2005 American Chemical Society

C O M M U N I C A T I O N S
M-1 min-1, and kmax/KArSH ) 4.05 × 106 M-1 min-1) induced from
the above data are almost identical with that of selenosubtilisin
(kmax/KH O ) 1.37 × 104 M-1 min-1, and kmax/KArSH ) 1.1 × 107
2
2
M-1 min-1).15 The observation revealed that the tellurosubtilisin
also favors the aromatic thiol substrate in the evolved specific
binding site of subtilisin. To further prove this, we compared the
catalytic efficiency of tellurosubtilisin with PhSeSePh in the
classical-coupled enzyme assay using GSH as a thiol substrate and
found that the former is only 8 times more efficient than the latter
for GPx activity.11
In summary, we have reengineered the active site of subtilisin
by chemical conversion of the catalytically essential serine into a
tellurocysteine and produced the semisynthetic telluroprotein,
tellurosubtilisin. It can catalyze the reduction of hydroperoxide by
an aryl thiol with high catalytic efficiency, but it exhibits kinetic
properties substantially different from those of selenosubtilisin. It
is anticipated that tellurosubtilisin will present an ideal model for
further studies of tellurium chemistry in protein.
Figure 2. Lineweaver-Burk plots for reduction of H2O2 by ArSH catalyzed
by 1 µM tellurosubtilisin at 25 °C and pH 5.5 (100 mM MES buffer, 10
mM CaCl2, 1 mM EDTA) at various concentrations of the substrate H2O2:
(1) 1.0 mM, (b) 2.0 mM, (2) 4.0 mM, (9) 8.0 mM, and (O) 25.0 mM.
Each point was measured in triplicate, and standard error was less than
5%.
gauge the catalytic ability of tellurosubtilisin, we compared its
catalytic efficiency with that of selenosubtilisin and diphenyl
diselenide (PhSeSePh). At 25 °C and pH 5.5, the initial rates for
the reduction of H2O2 (250 µM) by ArSH (100 µM) in the presence
of 1 µM enzyme are (3.9 ( 0.2) × 10-6 and (2.1 ( 0.1) × 10-6
M min-1 for tellurosubtilisin and selenosubtilisin, respectively.
Under similar conditions, but with 500 µM diphenyl diselenide as
Acknowledgment. We are grateful for the financial support of
973 Project (G2000078102), 863 Project (2004AA215250), NSFC
(20471023), and the Program for Changjiang Scholars and Innova-
tive Research Team in University.
the catalyst, the initial rate is only (0.95 ( 0.05) × 10-7 M min-1
.
Supporting Information Available: Synthesis and full character-
ization of the tellurosubtilisin, experimental details for measurements
of the GPx catalytic activity and hydrolytic activity, and for the
inhibition of GPx activity. This material is available free of charge via
the Internet at http://pubs.acs.org.
Thus, under the above conditions, the tellurosubtilisin was ap-
proximately 2 times and 20 000 times more efficient than seleno-
subtilisin and PhSeSePh for GPx activity. The maximal catalytic
rate of tellurosubtilisin was observed below pH 6.0.11 Furthermore,
importantly, the tellurosubtilisin, stored in PIPES buffer (pH 7.0)
at 4 °C, was particularly stable, and its GPx activity remained
unchanged for several months.
References
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The kinetic experiments were carried out in MES buffer (pH
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subtilisin BPN′,13 rather than the ping-pong mechanism of native
GPx,1a indicating the formation of a ternary complex between
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V0
kmax[ArSH][H2O2]
)
[E] KArSH H O + KArSH[H2O2] + KH O [ArSH] + [ArSH][H2O2]
2
2
2 2
where V0 is the initial rate of the enzymatic reaction, and [E] stands
for the total concentration of the enzyme; kmax is a pseudo-first-
order rate constant, and KArSH and KH O are the Michaelis constants
2
2
for ArSH and H2O2, respectively. The constant, KArSH H O , has no
2
2
easily grasped physical meaning. From the plots, we can get kmax
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(11) For the details, see Supporting Information.
) 518.1 ( 23.2 min-1, KH O ) 20.9 ( 1.1 mM, KArSH ) 128 (
2
2
6 µM, and K
) 0.596 ( 0.031 mM2. The kmax value
ArSH H2O2
represents the turnover number, about 500 molecules of H2O2
degraded per minute per molecule of tellurosubtilisin saturated with
substrates. The KH O and KArSH values of tellurosubtilisin obtained
(12) Cavallini, D.; Graziani, M. T.; Dupre, S. Nature 1966, 212, 294.
(13) Peterson, E. B.; Hilvert, D. Biochemistry 1995, 34, 6616.
2
2
are obviously smaller than that of selenosubtilisin (KH O > 0.3 M
2
2
(14) (a) Cleland, W. W. AdV. Enzymol. 1977, 45, 273. (b) Rudolph, F. B.;
and KArSH > 400 µM).15 Although the kmax for tellurosubtilisin is
apparently lower than that for selenosubtilisin (kmax > 8000
min-1),15 the second-order rate constants (kmax/KH O ) 2.48 × 104
Fromm, H. J. Methods Enzymol. 1979, 63, 138.
(15) Bell, I. M.; Hilvert, D. Biochemistry 1993, 32, 13969.
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