Vol. 168, No. 3, 1990 May 16, 1990
AND BIOPHYSICAL RESEARCH COMMUNICATIONS Pages 944-951
BIOCHEMICAL
COMPLETE ANINO ACID SEQUNNCE OF YEAST TNIOLTRAN8FNRABE (QLUTARNDOXIN) John Gan*, Mark A. Polokoff, and Mohinder K. Sardana
Zhong-Ru
Department of Biological Merck Sharp & Dohme Research West Point, Pennsylvania Received
March
W. Jacobs'
Chemistry Laboratories 19486
7, 1990
The amino acid sequence of a thioltransferase isolated from Sa cch a romvces cerevisia? was determined. The protein was cleaved by trypsin, StaDhVlOCOCCUS aureug V8 protease, and cyanogen bromide. The peptides generated were purified by reverse phase HPLC. Sequencing of intact protein and its fragments were achieved by automated Edman degradation. The protein contains 106 amino Yeast thioltransferase showed acid residues with two cysteines. 51% structural similarity to pig liver thioltransferase and 34% to E. coli. glutaredoxin. 01990 Academic Press, Inc. Thioltransferases known to
catalyze
nonprotein
ornithine
supporting glutathione glutathione,
the
study
called
glutathione
have
presence
of thioredoxin glutaredoxin
the
reductase,
of
ribonucleotide
electrons
to glutaredoxin,
can regulate
enzyme
of reduced
was found
reduction
(l-4).
reported
deficient
from and finally
and
as a reductant
status
iodothyronine
been
944
It
(5).
For
5'-deiodonase, to
and
be activated
glutathione mutants
to be fully (10).
capable Thus
NADPH can flow to the
by (M-9).
of E.
coli,
a
of coupled
with
to
essential
*To whom correspondence should be addressed. 'Current address: Department of Biological chemistry, College of Medicine, University of California, Irvine, 0004-291x/90 $1.50 Copyright 0 1990 by Academic Press, Inc. All righfs of reproduction in any form reserved.
proteins
disulfides
thiol-disulfide
papain,
the
ubiquitous
of protein
their
kinase,
in
apparently
thioltransferase
decarboxylase
thioltransferase
protein
that
by modulating pyruvate
small,
reduction using
suggested
activities
During
the
disulfides
has been
example,
are
California CA 92717.
Vol.
168,
No.
3, 1990
sulfhydryl final
BIOCHEMICAL
groups
steps
in
the
reduction
suggests
possibility
that
is
from
The proteins
than
exists
between
E.
coli
E.
both
about
procaryotic
and higher
it
(15)
this
amino with
acid
F. coli
based
and mammalian
eukaryotic sequence
structures
(11-13).
determined
contain
This
for cells
(13,
an active
16,
site,
Cys-
sequences
a 31% structure (18).
show similarity
Although
yeast
glutathione-homocystine
20 years is
also
(14).
proteins
called
protein
review
the
reductive
and primary
only
out
on catalytic
thioltransferase
However,
carried
the
and mammalian
sources
was described
information
the
coli
originally
transhydrogenase
catalyzes
same proteins
been
The mammalian
thioltransferase,
compare
have
85% similarity.
describe
the
COMMUNICATIONS
Glutaredoxin
Evidence
in a recent
from
Pro-Phe(Tyr)-Cys.
which
are
structures
thioltransferases
more
they
which
ribonucleotides.
cross-reactivity,
discussed
The primary
RESEARCH
reductase
disulfides.
immunological
strongly
17).
of
activity
of a variety
properties,
BIOPHYSICAL
of ribonucleotide
has a thioltransferase cleavage
AND
ago
available, cells.
and mammalian
no structural
leaving In
of yeast
(19),
this
a gap between report,
thioltransferase
we and
proteins.
BXPBRIRBRTAL PROCBDURBS Materials-Yeast thioltransferase was prepared as described elsewhere. TPCK treated trypsin was purchased from Sigma. Stanhvlococcus aureus V8 protease was from Pierce. A reverse phase Cl8 HPLC column was purchased from Vydac. All the other reagents were either HPLC or analytical grade. Carboxymethylation-The reduced protein was carboxymethylated by iodoacetamide in the presence of 6 M guanidine-HCl and 50 rat4 TrisHCl, pH 8.8. The excess iodoacetamide was removed by reverse phase HPLC. Bnrymatio cleavage of oarboxymethylated protein-All the proteolytic reactions were carried out in 0.2 M ammonium bicarbonate, pH 7.9, at 37' C. The reactions proceeded for 6 h at a protein to enzyme ratio of 60 (w/w). The resulting peptides were purified by reverse phase HPLC after lyophilization. Cyanogen bromide cleavage-The reaction was carried in 1 M cyanogen bromide in the presence of 70% formic acid for 24 h at room Excess cyanogen bromide was removed by speed vacuum temperature. centrifugation. Peptide isolation-All peptides were purified on reverse phase Cl8 HPLC with an acetonitrile gradient in the presence of 0.1% trifluoroacetic acid.
945
Vol.
BIOCHEMICAL
168, No. 3, 1990
AND BIOPHYSICAL
RESEARCH COMMUNlCATlONS
Amino aaid composition analysis-The peptides were hydrolyzed with constant boiling HCl in sealed evacuated reaction vials at 110' C for 20 hours. Amino acids were determined as phenylisothiocyanate (PITC) derivatives. Amino aoid sequence analysis-Sequence analysis was performed by automated Edman degradation on an Applied Biosystem 477A Protein Sequencer. The procedures used were from the manufacture. RESULTS The amino with
the
are
shown
acid
sequence
alignment in
of
Fig.
the
1.
of yeast
peptides
Direct
thioltransferase
used
sequencing
to
together
establish
of the
the
intact
sequence
protein
10 Val-Gor-Qln-Glu-Thr-Val-Ala-Ei~-Val-Lym-~p-Leu-Ilo-Gly-G1n-Lya-G1u-valCNBrl
II
I Vl
I
I
L-IL T-2
Tl 20 Phe-Val-Ala-Ala-Ly~-Thr-Tyr-Cy~-Pro-Tyr-Cy~-Lys-Ala-Thr-Leu-8er-Thr-Leu-
I
30
I
I
T3
TI
40 Phe-Gln-alu-Leu-Asn-Val-Pro-Lys-Ger-Lys-Ala-Leu-Val-Leu-Qlu-Leu-Asp-GluI
,
50
1
I v3
I
, T!i
60 Met-Ger-Asn-Gly-Ger-Glu-Ile-Gln-A~p-Ala-Leu-Glu-Glu-Ile-8er-Gly-Gln-Lys-
70
Cmr2
1 VI
80 Thr-val-Pro-Aen-Val-Tyr-Ile-Asn-Gly-Lys-Eis-Ile-Gly-Gly-Aaa-Ger-Asp-L~u
90
T6 100
1,
1
vs
I
I T7
Fig.
1. Amino acid
ssguonao of yeast thioltrumferrsa. The from cleavage of yeast thioltransferase with (CNBr), S, aureya V8 protease (V), and trypsin (T) Numbers consecutively from the N-terminus of the intact molecule are given on the top of the sequence. peptides cyanogen are shown.
derived bromide
946
from
Vol.
168, No. 3, 1990
its
unblocked
to 50.
BIOCHEMICAL
N-terminus
that
the
protein
carboxymethylated Fractionation two peaks,
protein
corresponding sequencing
overlap
was cleaved
(data not shown). CNBr2
protease
peptides
sequenced.
the
Acid hydrolysis*
L43U Vr
Phe His
LYS Trp
Total molar ratio determined.
digestion
was
2 10 6 7 15 4 7 7 10 1 6 12 3 2 2 12 106
determined
and the
Staohvlococcus V8
phase HPLC were CNBrl and CNBr2.
the sequence by 6
of the protein
end
Protain
would
with
trypsin
Fragment
cmr2
CNBr2
Sequence
ND' 107
of
by
the protein.
protein
1.3 11.0 5.8 6.3 15.9 4.7 7.4 7.3 8.7 0.8 6.2 12.6 2.9 2.3 2.4 12.4
analysis
which
peptide.
CNBr2 and extended
of the Intaat
Intact
CM-cys Asx Thr Ser Glx Pro =Y Ala Val Met Ile
paptides
none of them overlapped
Further
composition
HPLC resolved
The amino acid sequence
was then used to cleave
(96-101).
the
of the CNBrl fragment
To obtain
V5 overlapped
protein
I),
of 56 to 95 was obtained
peptide.
Unfortunately,
Amino Acid
phase
from the 5 major peaks on reverse
However peptide residues
(Table
by reverse
CNBrl and CNBr2 and the C-terminal
gyreus V8 protease
intact
1
by cyanogen bromide.
it was a mixture
to the residues the
of
The amino acid composition
and CNBr2.
that
analysis
one methionine
of the CNBr peptides
CNBrl indicated intact
had only
protein
CNBrl
RESEARCH COMMUNICATIONS
gave the amino acid sequence of residues
Since the amino acid composition
showed
*The 'Not
AND BfOPHYSfCAL
after
941
Acid hydrolysis*
Sequence
6.8 1.9 3.6 7.2 1.8 6.0 2.1 2.7
7 2 4 7 2 6 2 3
4.6 4.9 0.9
5 5 1
1.1 6.3 ND’ 51
1 6
HCl hydrolysis
51
at 1lO'C for
20 h.
BIOCHEMICAL
Vol. 168, No. 3, 1990
gave
7 peptides.
peptides which
showed
that
overlapped
and it basic
The amino
was placed residue
at
at
composition
its
analysis
sequence
(data
contains
the
reported
to
compositions
not amino
sequence
peptide
and CNBl2. the
end
of this
peptide
acid
intact
T5 contained
C-terminus
shown).
It
(Fig.
the
sequence peptide
protein
lacked
1).
was also be noted
are
since
The amino
that
with
peptide
its T7
acid in
Table
I.
0 2: 3:
!i
TLFBELNVPKSKALVLELDEnSNOSEI---O----g,-~----~---~-------~--~RDDF~y~yv
Pig. 2. Alignment of amino aaid aoquana~m of yaast thioltransferase, pig liver thioltraasfarasa, and 3. aoli glutareboxin. The numbers in the Figure represent the protein sequences as follows: 1, pig liver thioltransferase; 2, Yeast thioltransferase: and 3, E. coli glutaredoxin. Panel A, sequence
comparison between pig liver thioltransferase and yeast thioltransferase and panel B, between yeast thioltransferase and L & glutaredoxin. The N-terminus of pig liver thioltransferase is a N-acetylated alanine. The gaps are indicated by dashed lines. The amino acid sequences which are identical are boxed.
948
a
has been
Amino given
V5
acid
consistent
which
cleavage.
and CNBr2
of these
T7 overlapped
-Lys-Pro-,
tryptic
protein
of the
should
sequence, to
analysis
Peptide
carboxyl
be resistant of
acid
tryptic
CNBrl
AND BIOPHYSICAL RESEARCH COMMUNICATIONS
The
Vol.
BIOCHEMICAL
168, No. 3, 1990
values
determined
from
the
by acid
thymus,
(13),
but
structures pig
liver,
less
in
with
glutaredoxin
with
those
calculated
CO.,
liver
analysis
sequence and pig
that
between
only
34%.
2).
yeast
yeast
highly
conserved
between
mammalian
introduces
gaps
The sequence
analyzed
Hitachi
similarity
with
Software
necessary
to between
is calculated
thioltransferase
To
coli
were
from
the
(15).
thioltransferase
and 5.
obtained
thioltransferase
from
glutaredoxin
The sequences
program
which
are
was observed
sequences,
similarity.
liver
marrow
thioltransferase (Fig.
LTD
(glutaredoxin)
and E.
these
separately
Engineering
bone
similarity
of
pig
sequence
maximize
and rabbit
comparison
was aligned
yeast
agreed
thioltransferase
(glutaredoxin)
the
a protein
of
structural
thioltransferase aid
hydrolysis
RESEARCH COMMUNICATIONS
sequence.
The primary calf
AND BIOPHYSICAL
as 51% while
and E. coli
glutaredoxin
is
DISCUSSIOI Yeast cysteines more
thioltransferase and no arginine
similar
to
glutaredoxin share
an active
aligning
residues.
mammalian 2).
dithiol
in
active
side.
site
active
group
It
site
on the proteins,
structure tripeptide conserved
site
mammalian
in
the
E.
than
sequences, amino
the
with
amino
proposed to
that the
cysteine
E.
thioltransferases region.
Ile-Gly-Gly-, coli
between
and yeast 949
proteins
is
coli all
sources
-Cys-Pro-Tyr(Phe)-Cys-. obtained the acid
presence
residues
an basic
Unlike have
the
the
acid
yeast
a second
near
and ]Et dithiol the
cysteines
although
C-
sulfhydryl
Interestingly, these
by of
at
amino
low pKa of the
(20).
two
sequence
from
indicates
basic
at C-terminal
sequence,
acid
amino
tetrapeptide
may contribute
located
Its
thioltransferases
the
has been
active
acids
Cys-Pro-Tyr(Phe)-Cys-Xaa-Lys-,
one or two basic
terminal
106 amino
proteins
However
sequence, the
either
EeLi
the
(Fig.
A consensus
the
contains
lack
is the
BIOCHEMICAL
Vol. 168, No. 3, 1990
cysteine
residues.
on mammalian
protein
conserved of
It
region
interest
important
for
are
is
to
contrast,
the
blocked.
It
residue
of
thioltransferase,
cells,
was used.
hand,
the
to the
difference
in
is
activity.
The other
numbering).
It
two conserved
will
be
sequences
are
thioltransferases alanine
(13,
of the
enzymes
are
microbial that
to protect
acetylation
it (16).
lyophilized,
adding
any structural
N-terminal
During
the
preparation
of
dried
yeast,
instead
of
to be stable
inhibitors.
(1, the
not
by
protease
between
21).
degradation
seems
protein
of
16,
to against
displays
mammalian
acetylation
reflects
two cysteines
by an acetylated
thioltransferase
pH optimum
proteins
mammalian
speculated
without
yeast
enzyme
The thioltransferase
purification
additional
(yeast
and cathepsins
yeast
the
all
N-terminus
has been
aminopeptidases
for
of these
blocked
a protein
the
RESEARCH COMMUNICATIONS
function.
of are
essential
either
enzymatic
(glutaredoxins)
known if
-Thrn-Val-ProE-
know if
The N-terminus
In
is not
AND BtOPHYSlCAL
the 3, 4).
mammalian
or functional
through
On the
similar
fresh
other
kinetics
Whether
and
the
and microbial significance
is
unknown.
ACKNOWLBDGHEMTS We are their
invaluable
sequence Ii.
1. 2. 3. 4. 5. 6.
grateful
Waxman
to John assistance
and composition. for
critical
A. Rodkey in the
We thank reading
and Lori-Anne
determination Drs.
of this
William
of
T. Wassel the
W. Wells
amino
for acid
and Lloyd
manuscript.
Axelsson, K., Eriksson, S., and Mannervik, B. (1978) Biochemistry 17, 2978-2984. Axelsson, K., and Mannervik, B. (1980) Biochem. Biophys. Acta. 613, 324-336. Hatakeyama, M., Tanimoto, Y., and Mizoguchi, T. (1984) J. Biochem. 95, 1811-1818. Gan, Z-.R., and Wells, W.W. (1987) Anal. Biochem. 162, 265-273. Ziegler, D.M. (1985) Ann. Rev. Biochem. 54, 305-309. 152, 114-118. Axelsson, K., and Mannervik, B. (1983) FEB8 Lett. 950
Vol.
168, No.. 3, 1990
7.
Hatakeyama, M., Lee, C., Chon, C., Hayashi, M., and Mizoguchi, T. (1985) Biochem. Biophys. Res. Commu. 127, 458-463. Goswami, A., and Rosenberg, I.N. (1985) J. Biol. Chem. 260, 6012-6019. Flamigni, F., Marmiroli, S., Caldarera, C.M., and Guarnieri, C. (1989) Biochem. J. 259, 111-115. Holmgren, A. (1976) Proc. Natl. Acad. Sci. (USA) 73, 2275-2279. Luthman, M., and Holmgren, A. (1982) J. Biol. Chem. 257, 6686 -6690. Gan, Z-.R., and Wells, W.W. (1988) J. Biol. Chem. 263, 9051 -9054. Hopper, S., Johnson, R.S., Vath, J.E., and Biemann, K. (1989) J. Biol. Chem. 264, 20438-20447. Mannervik, B., Carlberg, I., and Larson, K. (1989) in Coenzymes and Cofactors (Dolphin, D., Poulson, R., and Avramovic, O., eds) Vol. 3, pp. 475-516, John Wiley & Sons, New York. H&g, J-.0., Jiirnvall, H., Holmgren, A., Carl&St, M., and Persson, M. (1983) Eur. J. Biochem. 136, 223-232. Gan, Z-.R., and Wells, W.W. (1987) J. Biol. Chem. 262, 6699 -6703. Papayannopoulos, I.A., Gan, Z-.R., Wells, W.W., and Biemann, K. (1989) Biochem. Biophys. Res. Commu. 159, 1448-1454. Klintrot, I-.M., Hodg, J-.0., Jornvall, H., Holmgren, A., and Luthman, M. (1984) Eur. J. Biochem. 144, 417-423. Nagai, S., and Black, S. (1968) J. Biol. Chem. 243, 1942-1947. Gan, Z-.R., and Wells, W.W. (1987) J. Biol. Chem. 262, 6704 -6707. Yang, Y., Gan, Z-.R., and Wells, W.W. (1989) Gene 83, 339-346.
8. 9. 10. 11. 12. 13. 14. 15. 16. 17. 18. 19. 20. 21.
BIOCHEMICAL
AND BIOPHYSICAL
951
RESEARCH COMMUNICATIONS
AND BIOPHYSICAL RESEARCH COMMUNICATIONS Pages 944-951
BIOCHEMICAL
COMPLETE ANINO ACID SEQUNNCE OF YEAST TNIOLTRAN8FNRABE (QLUTARNDOXIN) John Gan*, Mark A. Polokoff, and Mohinder K. Sardana
Zhong-Ru
Department of Biological Merck Sharp & Dohme Research West Point, Pennsylvania Received
March
W. Jacobs'
Chemistry Laboratories 19486
7, 1990
The amino acid sequence of a thioltransferase isolated from Sa cch a romvces cerevisia? was determined. The protein was cleaved by trypsin, StaDhVlOCOCCUS aureug V8 protease, and cyanogen bromide. The peptides generated were purified by reverse phase HPLC. Sequencing of intact protein and its fragments were achieved by automated Edman degradation. The protein contains 106 amino Yeast thioltransferase showed acid residues with two cysteines. 51% structural similarity to pig liver thioltransferase and 34% to E. coli. glutaredoxin. 01990 Academic Press, Inc. Thioltransferases known to
catalyze
nonprotein
ornithine
supporting glutathione glutathione,
the
study
called
glutathione
have
presence
of thioredoxin glutaredoxin
the
reductase,
of
ribonucleotide
electrons
to glutaredoxin,
can regulate
enzyme
of reduced
was found
reduction
(l-4).
reported
deficient
from and finally
and
as a reductant
status
iodothyronine
been
944
It
(5).
For
5'-deiodonase, to
and
be activated
glutathione mutants
to be fully (10).
capable Thus
NADPH can flow to the
by (M-9).
of E.
coli,
a
of coupled
with
to
essential
*To whom correspondence should be addressed. 'Current address: Department of Biological chemistry, College of Medicine, University of California, Irvine, 0004-291x/90 $1.50 Copyright 0 1990 by Academic Press, Inc. All righfs of reproduction in any form reserved.
proteins
disulfides
thiol-disulfide
papain,
the
ubiquitous
of protein
their
kinase,
in
apparently
thioltransferase
decarboxylase
thioltransferase
protein
that
by modulating pyruvate
small,
reduction using
suggested
activities
During
the
disulfides
has been
example,
are
California CA 92717.
Vol.
168,
No.
3, 1990
sulfhydryl final
BIOCHEMICAL
groups
steps
in
the
reduction
suggests
possibility
that
is
from
The proteins
than
exists
between
E.
coli
E.
both
about
procaryotic
and higher
it
(15)
this
amino with
acid
F. coli
based
and mammalian
eukaryotic sequence
structures
(11-13).
determined
contain
This
for cells
(13,
an active
16,
site,
Cys-
sequences
a 31% structure (18).
show similarity
Although
yeast
glutathione-homocystine
20 years is
also
(14).
proteins
called
protein
review
the
reductive
and primary
only
out
on catalytic
thioltransferase
However,
carried
the
and mammalian
sources
was described
information
the
coli
originally
transhydrogenase
catalyzes
same proteins
been
The mammalian
thioltransferase,
compare
have
85% similarity.
describe
the
COMMUNICATIONS
Glutaredoxin
Evidence
in a recent
from
Pro-Phe(Tyr)-Cys.
which
are
structures
thioltransferases
more
they
which
ribonucleotides.
cross-reactivity,
discussed
The primary
RESEARCH
reductase
disulfides.
immunological
strongly
17).
of
activity
of a variety
properties,
BIOPHYSICAL
of ribonucleotide
has a thioltransferase cleavage
AND
ago
available, cells.
and mammalian
no structural
leaving In
of yeast
(19),
this
a gap between report,
thioltransferase
we and
proteins.
BXPBRIRBRTAL PROCBDURBS Materials-Yeast thioltransferase was prepared as described elsewhere. TPCK treated trypsin was purchased from Sigma. Stanhvlococcus aureus V8 protease was from Pierce. A reverse phase Cl8 HPLC column was purchased from Vydac. All the other reagents were either HPLC or analytical grade. Carboxymethylation-The reduced protein was carboxymethylated by iodoacetamide in the presence of 6 M guanidine-HCl and 50 rat4 TrisHCl, pH 8.8. The excess iodoacetamide was removed by reverse phase HPLC. Bnrymatio cleavage of oarboxymethylated protein-All the proteolytic reactions were carried out in 0.2 M ammonium bicarbonate, pH 7.9, at 37' C. The reactions proceeded for 6 h at a protein to enzyme ratio of 60 (w/w). The resulting peptides were purified by reverse phase HPLC after lyophilization. Cyanogen bromide cleavage-The reaction was carried in 1 M cyanogen bromide in the presence of 70% formic acid for 24 h at room Excess cyanogen bromide was removed by speed vacuum temperature. centrifugation. Peptide isolation-All peptides were purified on reverse phase Cl8 HPLC with an acetonitrile gradient in the presence of 0.1% trifluoroacetic acid.
945
Vol.
BIOCHEMICAL
168, No. 3, 1990
AND BIOPHYSICAL
RESEARCH COMMUNlCATlONS
Amino aaid composition analysis-The peptides were hydrolyzed with constant boiling HCl in sealed evacuated reaction vials at 110' C for 20 hours. Amino acids were determined as phenylisothiocyanate (PITC) derivatives. Amino aoid sequence analysis-Sequence analysis was performed by automated Edman degradation on an Applied Biosystem 477A Protein Sequencer. The procedures used were from the manufacture. RESULTS The amino with
the
are
shown
acid
sequence
alignment in
of
Fig.
the
1.
of yeast
peptides
Direct
thioltransferase
used
sequencing
to
together
establish
of the
the
intact
sequence
protein
10 Val-Gor-Qln-Glu-Thr-Val-Ala-Ei~-Val-Lym-~p-Leu-Ilo-Gly-G1n-Lya-G1u-valCNBrl
II
I Vl
I
I
L-IL T-2
Tl 20 Phe-Val-Ala-Ala-Ly~-Thr-Tyr-Cy~-Pro-Tyr-Cy~-Lys-Ala-Thr-Leu-8er-Thr-Leu-
I
30
I
I
T3
TI
40 Phe-Gln-alu-Leu-Asn-Val-Pro-Lys-Ger-Lys-Ala-Leu-Val-Leu-Qlu-Leu-Asp-GluI
,
50
1
I v3
I
, T!i
60 Met-Ger-Asn-Gly-Ger-Glu-Ile-Gln-A~p-Ala-Leu-Glu-Glu-Ile-8er-Gly-Gln-Lys-
70
Cmr2
1 VI
80 Thr-val-Pro-Aen-Val-Tyr-Ile-Asn-Gly-Lys-Eis-Ile-Gly-Gly-Aaa-Ger-Asp-L~u
90
T6 100
1,
1
vs
I
I T7
Fig.
1. Amino acid
ssguonao of yeast thioltrumferrsa. The from cleavage of yeast thioltransferase with (CNBr), S, aureya V8 protease (V), and trypsin (T) Numbers consecutively from the N-terminus of the intact molecule are given on the top of the sequence. peptides cyanogen are shown.
derived bromide
946
from
Vol.
168, No. 3, 1990
its
unblocked
to 50.
BIOCHEMICAL
N-terminus
that
the
protein
carboxymethylated Fractionation two peaks,
protein
corresponding sequencing
overlap
was cleaved
(data not shown). CNBr2
protease
peptides
sequenced.
the
Acid hydrolysis*
L43U Vr
Phe His
LYS Trp
Total molar ratio determined.
digestion
was
2 10 6 7 15 4 7 7 10 1 6 12 3 2 2 12 106
determined
and the
Staohvlococcus V8
phase HPLC were CNBrl and CNBr2.
the sequence by 6
of the protein
end
Protain
would
with
trypsin
Fragment
cmr2
CNBr2
Sequence
ND' 107
of
by
the protein.
protein
1.3 11.0 5.8 6.3 15.9 4.7 7.4 7.3 8.7 0.8 6.2 12.6 2.9 2.3 2.4 12.4
analysis
which
peptide.
CNBr2 and extended
of the Intaat
Intact
CM-cys Asx Thr Ser Glx Pro =Y Ala Val Met Ile
paptides
none of them overlapped
Further
composition
HPLC resolved
The amino acid sequence
was then used to cleave
(96-101).
the
of the CNBrl fragment
To obtain
V5 overlapped
protein
I),
of 56 to 95 was obtained
peptide.
Unfortunately,
Amino Acid
phase
from the 5 major peaks on reverse
However peptide residues
(Table
by reverse
CNBrl and CNBr2 and the C-terminal
gyreus V8 protease
intact
1
by cyanogen bromide.
it was a mixture
to the residues the
of
The amino acid composition
and CNBr2.
that
analysis
one methionine
of the CNBr peptides
CNBrl indicated intact
had only
protein
CNBrl
RESEARCH COMMUNICATIONS
gave the amino acid sequence of residues
Since the amino acid composition
showed
*The 'Not
AND BfOPHYSfCAL
after
941
Acid hydrolysis*
Sequence
6.8 1.9 3.6 7.2 1.8 6.0 2.1 2.7
7 2 4 7 2 6 2 3
4.6 4.9 0.9
5 5 1
1.1 6.3 ND’ 51
1 6
HCl hydrolysis
51
at 1lO'C for
20 h.
BIOCHEMICAL
Vol. 168, No. 3, 1990
gave
7 peptides.
peptides which
showed
that
overlapped
and it basic
The amino
was placed residue
at
at
composition
its
analysis
sequence
(data
contains
the
reported
to
compositions
not amino
sequence
peptide
and CNBl2. the
end
of this
peptide
acid
intact
T5 contained
C-terminus
shown).
It
(Fig.
the
sequence peptide
protein
lacked
1).
was also be noted
are
since
The amino
that
with
peptide
its T7
acid in
Table
I.
0 2: 3:
!i
TLFBELNVPKSKALVLELDEnSNOSEI---O----g,-~----~---~-------~--~RDDF~y~yv
Pig. 2. Alignment of amino aaid aoquana~m of yaast thioltransferase, pig liver thioltraasfarasa, and 3. aoli glutareboxin. The numbers in the Figure represent the protein sequences as follows: 1, pig liver thioltransferase; 2, Yeast thioltransferase: and 3, E. coli glutaredoxin. Panel A, sequence
comparison between pig liver thioltransferase and yeast thioltransferase and panel B, between yeast thioltransferase and L & glutaredoxin. The N-terminus of pig liver thioltransferase is a N-acetylated alanine. The gaps are indicated by dashed lines. The amino acid sequences which are identical are boxed.
948
a
has been
Amino given
V5
acid
consistent
which
cleavage.
and CNBr2
of these
T7 overlapped
-Lys-Pro-,
tryptic
protein
of the
should
sequence, to
analysis
Peptide
carboxyl
be resistant of
acid
tryptic
CNBrl
AND BIOPHYSICAL RESEARCH COMMUNICATIONS
The
Vol.
BIOCHEMICAL
168, No. 3, 1990
values
determined
from
the
by acid
thymus,
(13),
but
structures pig
liver,
less
in
with
glutaredoxin
with
those
calculated
CO.,
liver
analysis
sequence and pig
that
between
only
34%.
2).
yeast
yeast
highly
conserved
between
mammalian
introduces
gaps
The sequence
analyzed
Hitachi
similarity
with
Software
necessary
to between
is calculated
thioltransferase
To
coli
were
from
the
(15).
thioltransferase
and 5.
obtained
thioltransferase
from
glutaredoxin
The sequences
program
which
are
was observed
sequences,
similarity.
liver
marrow
thioltransferase (Fig.
LTD
(glutaredoxin)
and E.
these
separately
Engineering
bone
similarity
of
pig
sequence
maximize
and rabbit
comparison
was aligned
yeast
agreed
thioltransferase
(glutaredoxin)
the
a protein
of
structural
thioltransferase aid
hydrolysis
RESEARCH COMMUNICATIONS
sequence.
The primary calf
AND BIOPHYSICAL
as 51% while
and E. coli
glutaredoxin
is
DISCUSSIOI Yeast cysteines more
thioltransferase and no arginine
similar
to
glutaredoxin share
an active
aligning
residues.
mammalian 2).
dithiol
in
active
side.
site
active
group
It
site
on the proteins,
structure tripeptide conserved
site
mammalian
in
the
E.
than
sequences, amino
the
with
amino
proposed to
that the
cysteine
E.
thioltransferases region.
Ile-Gly-Gly-, coli
between
and yeast 949
proteins
is
coli all
sources
-Cys-Pro-Tyr(Phe)-Cys-. obtained the acid
presence
residues
an basic
Unlike have
the
the
acid
yeast
a second
near
and ]Et dithiol the
cysteines
although
C-
sulfhydryl
Interestingly, these
by of
at
amino
low pKa of the
(20).
two
sequence
from
indicates
basic
at C-terminal
sequence,
acid
amino
tetrapeptide
may contribute
located
Its
thioltransferases
the
has been
active
acids
Cys-Pro-Tyr(Phe)-Cys-Xaa-Lys-,
one or two basic
terminal
106 amino
proteins
However
sequence, the
either
EeLi
the
(Fig.
A consensus
the
contains
lack
is the
BIOCHEMICAL
Vol. 168, No. 3, 1990
cysteine
residues.
on mammalian
protein
conserved of
It
region
interest
important
for
are
is
to
contrast,
the
blocked.
It
residue
of
thioltransferase,
cells,
was used.
hand,
the
to the
difference
in
is
activity.
The other
numbering).
It
two conserved
will
be
sequences
are
thioltransferases alanine
(13,
of the
enzymes
are
microbial that
to protect
acetylation
it (16).
lyophilized,
adding
any structural
N-terminal
During
the
preparation
of
dried
yeast,
instead
of
to be stable
inhibitors.
(1, the
not
by
protease
between
21).
degradation
seems
protein
of
16,
to against
displays
mammalian
acetylation
reflects
two cysteines
by an acetylated
thioltransferase
pH optimum
proteins
mammalian
speculated
without
yeast
enzyme
The thioltransferase
purification
additional
(yeast
and cathepsins
yeast
the
all
N-terminus
has been
aminopeptidases
for
of these
blocked
a protein
the
RESEARCH COMMUNICATIONS
function.
of are
essential
either
enzymatic
(glutaredoxins)
known if
-Thrn-Val-ProE-
know if
The N-terminus
In
is not
AND BtOPHYSlCAL
the 3, 4).
mammalian
or functional
through
On the
similar
fresh
other
kinetics
Whether
and
the
and microbial significance
is
unknown.
ACKNOWLBDGHEMTS We are their
invaluable
sequence Ii.
1. 2. 3. 4. 5. 6.
grateful
Waxman
to John assistance
and composition. for
critical
A. Rodkey in the
We thank reading
and Lori-Anne
determination Drs.
of this
William
of
T. Wassel the
W. Wells
amino
for acid
and Lloyd
manuscript.
Axelsson, K., Eriksson, S., and Mannervik, B. (1978) Biochemistry 17, 2978-2984. Axelsson, K., and Mannervik, B. (1980) Biochem. Biophys. Acta. 613, 324-336. Hatakeyama, M., Tanimoto, Y., and Mizoguchi, T. (1984) J. Biochem. 95, 1811-1818. Gan, Z-.R., and Wells, W.W. (1987) Anal. Biochem. 162, 265-273. Ziegler, D.M. (1985) Ann. Rev. Biochem. 54, 305-309. 152, 114-118. Axelsson, K., and Mannervik, B. (1983) FEB8 Lett. 950
Vol.
168, No.. 3, 1990
7.
Hatakeyama, M., Lee, C., Chon, C., Hayashi, M., and Mizoguchi, T. (1985) Biochem. Biophys. Res. Commu. 127, 458-463. Goswami, A., and Rosenberg, I.N. (1985) J. Biol. Chem. 260, 6012-6019. Flamigni, F., Marmiroli, S., Caldarera, C.M., and Guarnieri, C. (1989) Biochem. J. 259, 111-115. Holmgren, A. (1976) Proc. Natl. Acad. Sci. (USA) 73, 2275-2279. Luthman, M., and Holmgren, A. (1982) J. Biol. Chem. 257, 6686 -6690. Gan, Z-.R., and Wells, W.W. (1988) J. Biol. Chem. 263, 9051 -9054. Hopper, S., Johnson, R.S., Vath, J.E., and Biemann, K. (1989) J. Biol. Chem. 264, 20438-20447. Mannervik, B., Carlberg, I., and Larson, K. (1989) in Coenzymes and Cofactors (Dolphin, D., Poulson, R., and Avramovic, O., eds) Vol. 3, pp. 475-516, John Wiley & Sons, New York. H&g, J-.0., Jiirnvall, H., Holmgren, A., Carl&St, M., and Persson, M. (1983) Eur. J. Biochem. 136, 223-232. Gan, Z-.R., and Wells, W.W. (1987) J. Biol. Chem. 262, 6699 -6703. Papayannopoulos, I.A., Gan, Z-.R., Wells, W.W., and Biemann, K. (1989) Biochem. Biophys. Res. Commu. 159, 1448-1454. Klintrot, I-.M., Hodg, J-.0., Jornvall, H., Holmgren, A., and Luthman, M. (1984) Eur. J. Biochem. 144, 417-423. Nagai, S., and Black, S. (1968) J. Biol. Chem. 243, 1942-1947. Gan, Z-.R., and Wells, W.W. (1987) J. Biol. Chem. 262, 6704 -6707. Yang, Y., Gan, Z-.R., and Wells, W.W. (1989) Gene 83, 339-346.
8. 9. 10. 11. 12. 13. 14. 15. 16. 17. 18. 19. 20. 21.
BIOCHEMICAL
AND BIOPHYSICAL
951
RESEARCH COMMUNICATIONS
