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INTRODUCTION |
The glycine cleavage system is a multienzyme complex composed of
four proteins, P-, H-, T-, and L-protein, which catalyzes the
reversible oxidation of glycine yielding carbon dioxide, ammonia, 5,10-CH2-H4folate,1
and a reduced pyridine nucleotide. T-protein is a
folate-dependent enzyme that catalyzes the release of
ammonia and the transfer of the methylene carbon unit to
H4folate from the intermediate attached to H-protein after
the decarboxylation of glycine catalyzed by P-protein (reviewed in Ref.
1). The kinetic properties of the forward (2) and the reverse (3)
reaction catalyzed by T-protein have been studied extensively, and the
primary structures of T-proteins from eight species have been
determined (4-10) as well as many putative T-protein sequences derived
from genome analyses.
We found previously that lysine residues of Escherichia coli
T-protein (ET) at positions 78, 81, and 352 are involved in the interaction with the polyglutamate moiety of
5,10-CH2-H4PteGlu4 (11). From the
results of a kinetic analysis with single and multiple substitution
mutants for these lysine residues, it was postulated that
Lys352 interacts with the
-carboxyl group of the first
glutamate residue nearest the p-aminobenzoic acid ring of
5,10-CH2-H4PteGlu4 and that
Lys81 interacts with the
-carboxyl group of the second
glutamate residue. Lys78 seems to participate in the
binding of the peripheral glutamate residues. Lys352 is
conserved among the T-proteins studied thus far, whereas
Lys81 and Lys78 are not. Therefore, other
lysine residues must be responsible for the binding of the second and
third glutamate residue of
5,10-CH2-H4PteGlu4 in T-proteins
from other species. Furthermore, a cross-linking study (12) revealed
that the interaction of E. coli H-protein (EH) with ET
causes a conformational change of ET and results in intramolecular
cross-linking between Asp34 and Lys216 of ET.
Intermolecular cross-linking between Lys288 of ET and
Asp43 of EH was also identified, indicating the
participation of the region in the interaction between ET and EH. The
extreme N-terminal region of ET is essential for the interaction with
EH because such intramolecular and intermolecular cross-linking was not
observed in a complex composed of the N-terminal 7-residue deletion
mutant of ET (ET
7) and EH. The marked decrease in the
kcat/Km value of ET
7 for
EH compared with the wild type enzyme exhibited a good correlation with
the cross-linking results.
In the present study, we used limited proteolysis to further examine
the function of the N-terminal region as well as the mobile nature of
the T-protein catalysis. Two amino acid residues, Thr4 and
Leu6, that are conserved in all eight T-proteins were
mutated separately to alanine. The resulting mutants (ETT4A and ETL6A),
ET, and ET
7 were overexpressed as His6-tagged proteins
at the C-terminal end, purified, and subjected to proteolysis. The
profile of protease digestion of these proteins in the absence or
presence of substrates suggested that the functional lysine residues of
ET identified previously are protease-accessible and that some of them
are buried in the ET-EH complex. Moreover, it is revealed that the
N-terminal region serves as a molecular "hasp" to hold the ET
molecule in the proper folding critical for association with EH.
Leu6 plays a pivotal role in the hasp function. The
formation of the folate binding cavity by the interaction of ET with EH
was also probed by limited proteolysis.
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EXPERIMENTAL PROCEDURES |
Materials--
[14C]Sodium bicarbonate was
obtained from Amersham Biosciences. Restriction endonucleases and other
DNA modifying enzymes were purchased from New England Biolabs, Roche
Molecular Biochemicals, Toyobo (Tokyo, Japan), or Takara Shuzo (Shiga,
Japan). Oligonucleotides were from Hokkaido System Science (Sapporo,
Japan). L-1-Tosylamido-2-phenylethyl chloromethyl
ketone-treated bovine pancreatic trypsin (TPCK-trypsin) was purchased
from Worthington, and soybean trypsin inhibitor was from Sigma.
Lysylendopeptidase (Lys-C) and folic acid were obtained from Wako Pure
Chemicals (Osaka, Japan). Pteroyltetraglutamate from Schircks
Laboratory (Jona, Switzerland) and folic acid were reduced by the
method of Kisliuk (13) and used for the synthesis of
5,10-CH2-H4PteGlu4 and
5,10-CH2-H4folate, respectively, as described
(14). Recombinant E. coli P-protein and H-protein were
prepared as described previously (11).
Expression and Purification of Wild Type and Mutant E. coli
T-protein with a His6 Tag at the C-terminal End--
DNA
manipulations were accomplished using standard techniques (15). To
construct plasmids for the expression of ET and ET
7 with a
His6 tag at the C-terminal end, pET3a with an insertion of
ET DNA (pET/ET) (11) or ET
7 DNA (12) was cut with EcoRI and employed in a polymerase chain reaction with
oligonucleotides 5'-TAATACGACTCACTATAGGG-3' (T7 promoter primer) as
5'-primer and 5'-AAAAGTCTCGAGCGCGACGGCTTTGCCG-3' (the
XhoI site is underlined) as 3'-primer. The products were
purified with a QIAEX II kit (Qiagen) after agarose gel
electrophoresis, digested with NdeI and XhoI, and
ligated into pET23b. The nucleotide sequences of the resultant plasmids, pET23b/ET and pET23b/ET
7, were confirmed with a 373 DNA
sequencing system (Applied Biosystems). For the introduction of two
mutations, ETT4A and ETL6A, polymerase chain reaction was carried out
using EcoRI-digested pET/ET as a template with
the respective 5'-primers,
5'-AGATATACATATGGCACAACAGGCTCCTTTG-3' and 5'-
AGATATACATATGGCACAACAGACTCCTGCGTACGAAC-3' (modified bases
are shown in boldface letters), and the common 3'-primer, 5'-AAAAGTCTCGAGCGCGACGGCTTTGCCG-3' (the
XhoI site is underlined). The products were subcloned into
pET23b as described above, and nucleotide sequences of the resulting
plasmids, pET23b/ETT4A and pET23b/ETL6A, were verified by DNA sequencing.
E. coli BL21(DE3)pLysS cells (16) transformed with the
constructed expression plasmids were grown in 100 ml of LB medium containing 20 µg/ml ampicillin and 25 µg/ml chloramphenicol at 30 °C for 24 h. Expression was induced by 25 µM
isopropyl-
-D-thiogalactopyranoside added at the start of
the incubation. Cell-free extracts were prepared as described
previously (7) with buffer A (20 mM potassium phosphate, pH
7.4, 0.3 M NaCl, and 0.2 mM dithiothreitol)
containing 20 mM imidazole and subjected to affinity
chromatography in a Ni2+ column (1.5 × 2.2 cm,
Qiagen). The samples were eluted with buffer A containing 50 mM imidazole, concentrated with a Centriplus-30 (Millipore), and subjected to size exclusion chromatography on a
Superdex 200 pg 16/60 column equilibrated with 50 mM
potassium phosphate, pH 7.0, containing 0.2 M NaCl and 1 mM dithiothreitol. The final preparations were dialyzed
against 20 mM potassium phosphate, pH 7.0, containing 1 mM dithiothreitol, concentrated with a Centricon-10 (Millipore), and stored at
80 °C.
Protease Digestion and Analysis of the Fragments--
ETs (1.8 mg/ml) were digested in 50 mM HEPES, pH 7.0, at 25 °C
with TPCK-trypsin at an ETs/trypsin (w/w) ratio of 100:1. Aliquots containing 18 µg of ETs were removed at various times, and the digestion was terminated with a 2-fold excess (w/w) of soybean trypsin
inhibitor. When the trypsinolysis was carried out in the presence of
substrates, ETs were preincubated with a 2-fold molar excess of EH or
with 0.2 mM
5,10-CH2-H4PteGlu4 at 25 °C for
30 min in 50 mM HEPES, pH 7.0. Then TPCK-trypsin was added
at a ratio of 10:1 (w/w) and digested as above. In all
cases, an equal aliquot was withdrawn just before the addition of
TPCK-trypsin and used as a zero time control. Samples were subjected to
an assay of T-protein activity (12) and SDS-PAGE according to the
method of Schägger and von Jagow (17). Digestion by Lys-C was
also carried out as above in 20 mM Tris-HCl, pH 8.0, at an
ET/Lys-C ratio (w/w) of 50:1. In this case, the reaction was terminated by the addition of an equal volume of 2× SDS-PAGE sample buffer to
aliquots and immediate treatment in boiling water.
The proteolytic fragments were separated by SDS-PAGE, transferred to a
polyvinylidene difluoride membrane (Immobilon-PSQ,
Millipore), and subjected to Edman sequencing on a Hewlett Packard G100A protein sequencer. When necessary, the digestion products were
separated by reversed-phase high performance liquid chromatography as
described previously (11), and appropriate peak fractions were
subjected to N-terminal sequencing.
Assay of T-protein Activity and Kinetic Analysis--
The
routine assay and kinetic analysis of T-protein were conducted as
described previously (12). For the kinetic analysis of ET, ETT4A, and
ETL6A, concentrations of varying substrates were: reduced EH, 0.4-3.6
µM;
L-(±)-5,10-CH2-H4folate, 40-360 µM;
L-(±)-5,10-CH2-H4PteGlu4,
4-36 µM; NH4Cl, 20-180 mM; and
the concentrations of constant substrates were: reduced EH, 6 µM; L-(±)-5,10-CH2-H4folate, 0.6 mM; NH4Cl, 200 mM. In the
experiments employing ET
7, concentrations of varying substrates
were: reduced EH, 2.67-24 µM;
L-(±)-5,10-CH2-H4folate, 40-360
µM;
L-(±)-5,10-CH2-H4PteGlu4, 20-180 µM; NH4Cl, 30-300 mM;
and the concentrations of constant substrates were: reduced EH, 36 µM;
L-(±)-5,10-CH2-H4folate, 0.6 mM; NH4Cl, 300 mM. The experiments
determining the Km values for reduced EH of ET,
ETT4A, and ETL6A were conducted in 0.5 ml of reaction mixture.
Appropriate amounts of ETs within the following ranges were used: ET,
10-20 ng; ET
7, 80-250 ng; ETT4A, 20-40 ng; ETL6A, 20 ng.
Mass Spectrometry--
The tryptic fragments were analyzed by
matrix-assisted laser desorption ionization/time-of-flight mass
spectrometry. ET and ET·His6 (each 360 µg) digested
with TPCK-trypsin (36 µg) in the presence of a 2 molar excess
of EH at 25 °C for 90 min were loaded onto a Superdex 200 HR 10/30
column (Amersham Biosciences) equilibrated with 50 mM
Tris-HCl, pH 8.0, 0.2 M NaCl, and 1 mM
dithiothreitol. Two main peaks corresponding to molecular masses of 54 and 26 kDa were obtained. The former peak, containing a 39-kDa band
verified by SDS-PAGE, was concentrated using Microcon-3 (Millipore),
and the solvent was replaced with 50% acetonitrile containing 0.1% trifluoroacetic acid by repeated concentration. The sample solution was
mixed with a saturated solution of sinapinic acid
(3,5-dimethoxy-4-hydroxycinnamic acid) in 30% acetonitrile and 0.1%
trifluoroacetic acid. The spectra of positive ions were recorded in the
linear mode on a Voyager-DE STR mass spectrometer (Perseptive
Biosystems) equipped with a delayed extraction device. External
calibration was performed with Sequazyme Peptide Mass Standard Kit Mix
3 (Perseptive Biosystems) containing insulin (bovine), thioredoxin
(E. coli), and apomyoglobin (horse) with a mass
(M + H+1) of 5734.59, 11,674.48, and 16,952.56 Da, respectively.
Other Methods--
Protein concentrations were routinely
determined by the method of Bradford (18) with bovine serum albumin as
a standard. Concentrations of purified wild type and mutant ETs with a
His6 tag at the C terminus and EH were estimated as
described previously (11).
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RESULTS |
Expression and Purification of Wild Type and Mutant E. coli
T-protein with a His6 Tag at the C-terminal End--
To
facilitate purification and to obtain mutant ETs without contamination
by wild type ET originating from the host cell gene, we introduced a
His6 tag at the C-terminal end of the ETs with two extra
amino acids (-LEHHHHHH). Four proteins, termed ET·His6, ET
7·His6, ETT4A·His6, and
ETL6A·His6, were highly expressed in E. coli
as soluble forms and purified by Ni2+ affinity column
chromatography and subsequent gel filtration. The final preparation of
each protein gave a single band on SDS-PAGE (not shown). Purified
ET·His6 showed comparable specific activity (16,885 units/mg) to ET (15,127 units/mg) (12), indicating no significant
effect of the C-terminal extension on the activity. The specific
activity of purified ET
7·His6 (906 units/mg) was about
5% relative to ET·His6, again giving a result comparable to ET
7 (764 units/mg) (12). T4A and L6A mutations led to a reduction
in the specific activity to 43% (7221 units/mg) and 79% (13,364 units/mg), respectively, relative to wtET·His6.
Kinetic Analysis--
Steady state kinetic studies were carried
out by varying the concentration of one substrate and keeping the
concentrations of the other two substrates constant (Table
I). ET·His6 and
ET
7·His6 gave parameters comparable to those of ET and
ET
7, respectively (12), showing a notable decrease (165-fold) in the
kcat/Km value of
ET
7·His6 for reduced EH. ETT4A·His6 and
ETL6A·His6 also exhibited a significant decrease (4- and
5-fold, respectively) in the
kcat/Km value for reduced EH
compared with ET·His6. These results suggest that both
residues play an important role in the function of the N-terminal
region of ET.
Protease Digestion--
To investigate the effect of the
N-terminal mutation, the purified proteins were subjected to limited
proteolysis by TPCK-trypsin (an ETs/trypsin ratio of 100:1), and the
resulting peptides were analyzed by SDS-PAGE, Edman sequencing, assay
of the activity, and/or mass spectrometry. ET·His6 was
resistant to trypsin digestion as revealed by the presence of about
half of the intact ET band (Fig.
1A, lane 7) and the retention of about half of
the original activity (Fig. 3A) even after a 4-h
digestion. However, ET
7·His6 was quite
susceptible to trypsinolysis and converted to a 37-kDa fragment (F1)
within the first 5 min of digestion (Fig. 1B, lane 2). F1 gave the N-terminal sequence starting from
Met17 (Table II) and retained
the His6 tag at the C-terminal end as probed by the
antibody against the His6 tag sequence in Western blotting
(not shown). Loss of the N-terminal residues up to Arg16
resulted in a complete loss of activity (Fig. 3A). F1 was
rapidly degraded to small pieces through intermediary fragments of 20, 14, and 7 kDa (named F2, F3, and F4, respectively) (Fig. 1B,
lanes 2-5). Consequently, the cleavage at Arg16
seems to trigger the subsequent C-terminal fragmentation. The intermediate peptides were subjected to Edman sequencing, and the five
N-terminal residues were determined as
Met17-His21,
Ala82-Gly86, and
Met17-His21 for F2, F3, and F4, respectively
(Table II), predicting a split of F2 to F4 and F3. F2 and F3 probably
have a common C-terminal residue, which can be assumed as
Lys203 or Arg208 judging from the molecular
mass on SDS-PAGE. As several peptide bands found between F1 and F2
showed the same N-terminal sequence starting from Met17, it
is conceivable that random cleavage occurs at several positions in the
C-terminal one-third of ET
7·His6 after the removal of 16 residues from the N-terminal. ETL6A·His6 behaved
similar to ET
7·His6 (Fig. 1D) in
trypsinolysis, indicating a central role for the leucine residue in the
function of the N-terminal region. Arg16 of
ETT4A·His6 was somewhat resistant to trypsinolysis
(Fig. 1C).

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Fig. 1.
Time course of tryptic digestion of E. coli T-proteins. E. coli T-proteins were
digested with trypsin at an ET/trypsin (w/w) ratio of 100:1 as
described under "Experimental Procedures." Digests containing 3.3 µg of ET were loaded for SDS-PAGE (16.5% T and 3% C
acrylamide gels) (17) and stained with Coomassie Blue. A,
ET·His6; B, ET 7·His6;
C, ETT4A·His6; D,
ETL6A·His6. The digestion times in minutes are indicated
below the lanes, and the standard protein markers
(lanes M1 and M2) are on the left. The
fragments derived from ET 7·His6 are termed as
indicated on the right.
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The effects of substrate binding on susceptibility to trypsinolysis
were examined in the presence of EH (a 2-fold molar excess over ETs) or
5,10-CH2-H4PteGlu4. In these
experiments, TPCK-trypsin was employed at an ETs/trypsin (w/w) ratio of
10:1 to ensure the thorough digestion of ET·His6. As
shown in Fig. 2, both substrates exhibited negligible protection against the cleavage at
Arg16 of the mutants except for the partial protection of
ETT4A·His6 by EH. However, both substrates substantially
prevented the subsequent C-terminal fragmentation, although to somewhat
different extents. Digestion of ET·His6 by large amounts
of trypsin proceeded gradually without a remarkable accumulation of the
intermediate fragments except for the small amount of 30-kDa fragment,
which gave the N-terminal sequence starting from Ala1.
However, digestion of ET·His6 in the presence of EH
resulted in the accumulation of a 39-kDa fragment (F5) (Fig.
2B, lane 12). The fragment gave an intact
N-terminal amino acid sequence starting from Ala1 but did
not cross-react with the antibody against His6 tag (data not shown). Furthermore, gel-purified F5 gave a
[M + H+1] value of 39,767.52 Da, in agreement with the
calculated value for the peptide Ala1-Lys360
(39,773.13 Da). The equivalent peptide obtained from the parallel experiment with ET also gave an intact N-terminal sequence and a
[M + H+1] value of 39,771.85 Da, although it is
impossible to distinguish the mobility of the peptide band from that of
intact ET on SDS-PAGE (Fig. 2A, lane 12). In
contrast to the cleavage at Arg16, the cleavage at
Lys360 affected the activity only slightly (Fig.
3B). To further characterize the cleavage at Lys360, the tryptic digests of
ET·His6 in the absence or presence of EH were subjected
to reversed-phase HPLC. The peptide from Ala361 to the end
of the His6 tag (AVALEHHHHHH) was eluted at 8%
acetonitrile as an isolated peak with intensive absorbance at 220 nm.
The intensity was compared with that of the peptide
Met17-Arg40, a peptide reflecting the
fragmentation of the entire ET molecule into small pieces. The results
demonstrated that the release of the C-terminal peptide was
significantly stimulated in the presence of EH despite the marked
decrease of that of the peptide Met17-Arg40
(Fig. 4).
5,10-CH2-H4PteGlu4 also stimulated
the cleavage at Lys360 while suppressing the fragmentation
of the entire ET·His6, and EH potentiated the event (Fig.
4).

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Fig. 2.
The effects of substrates on the
trypsinolysis of E. coli T-proteins. E. coli T-proteins were digested with TPCK-trypsin at an ETs/trypsin
(w/w) ratio of 10:1 in the absence (lanes 1-6) or presence
(lanes 7-12) of a 2-fold molar excess of EH or 0.2 mM 5,10-CH2-H4PteGlu4
(lanes 13-18) as described under "Experimental
Procedures." The digests were analyzed as described in the legend for
Fig. 1. A, ET; B, ET·His6;
C, ET 7·His6; D,
ETT4A·His6; E, ETL4A·His6. The
digestion times in minutes are indicated below the lanes,
and each intact protein and the fragments derived from ET and
ET·His6 are indicated.
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Fig. 3.
T-protein activity during the tryptic
digestion. A, T-protein activity in the tryptic digests
analyzed in Fig. 1 was assayed as described under "Experimental
Procedures." B, ET·His6 digested with
TPCK-trypsin at an ET/trypsin (w/w) ratio of 10:1 in the absence or
presence of a 2-fold excess of EH was assayed.
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Fig. 4.
Detection of the cleavage at
Lys360 during trypsin digestion of
ET·His6. ET·His6 digests
treated with TPCK-trypsin in the absence or presence of EH and
CH2-H4PteGlu4 as described in Fig.
2 were subjected to reversed-phase HPLC. The intensities of the peaks
containing the C-terminal fragment
(Ala361-His6) or the fragment
Met17-Arg40 were compared after the
identification by Edman sequencing.
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In contrast to trypsin, Lys-C easily digested ET·His6 at
an ET·His6/Lys-C (w/w) ratio of 50:1, in which
several fragments accumulated as shown in Fig.
5. Edman sequencing of up to 5 residues of these fragments was carried out, and the C-terminal ends were predicted from the molecular masses determined by SDS-PAGE (Table II).
The results revealed that at least four lysine residues at positions of
81, 154, 288, and 360 are favorable cleavage sites. Cleavage at
Lys81 and Lys288 was strongly prohibited
in the presence of EH, although that at Lys360 was again
stimulated by the interaction with EH. About half of the EH that was
added, i.e. ET-bound EH, remained intact in contrast with
the immediate degradation of free EH.

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Fig. 5.
Time course of digestion of E. coli T-proteins by lysylendopeptidase.
ET·His6 was digested with Lys-C at an ET/Lys-C (w/w)
ratio of 50:1 in the absence (lanes 1-6) or presence
(lanes 7-12) of a 2-fold molar excess of EH as
described under "Experimental Procedures." The digestion times
in minutes are indicated below the lanes, and the standard
protein markers (lanes M1 and M2) are
indicated on the left. The fragments derived from
ET·His6 (termed F5-F12) and EH
(termed EH2 and EH3) are indicated on the
right.
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 |
DISCUSSION |
In this study, we used limited proteolysis and mutation analysis
to further elucidate the structural nature of ET in the catalytic reaction as well as the function of its N-terminal region in the interaction with EH. The mutant proteins were constructed by replacing two invariant residues among the 7 N-terminal residues,
Thr4 and Leu6, with alanine as well as
the deletion of up to 7 residues. The introduction of a
His6 tag to the C-terminal end not only facilitated the
purification of proteins having activity comparable with that of
proteins without His6 but also provided a convenient tool
for detecting local conformational change at the C terminus, as
mentioned below. A single mutation, T4A or L6A, resulted in a 4- or
5-fold decrease in the
kcat/Km value for reduced EH,
respectively, compared with ET, indicating the importance of these
residues in the T-protein activity.
The purified wild type and mutant proteins were subjected to limited
proteolysis. The difference in the profile of digestion of
ET·His6 and ET
7·His6 by a small amount
of TPCK-trypsin demonstrated that Arg16 in
ET
7·His6 is oriented toward a trypsin-accessible
location. After cleavage at Arg16 and loss of 16 N-terminal
residues, the remaining C-terminal polypeptide chain would be unfolded
and attacked by trypsin. Because EH and folate substrate substantially
prevented the latter event, it is conceivable that they facilitate the
intramolecular interaction between the relatively stable
N-terminal two-thirds and the extended C-terminal one-third of ET.
These findings are consistent with our previous suggestion that
Lys352 serves as the binding site for the first glutamate
residue nearest the p-aminobenzoic acid ring of
5,10-CH2-H4PteGlu4 and that
Lys81 and Lys78 participate in the binding with
the second and third glutamate residue, respectively (11).
Consequently, the N-terminal region might serve as the molecular hasp
locking its own molecule in a compact fold. The point mutations, L6A
and T4A, also resulted in an extended N terminus, probably because of
the loss of interaction between these residues and some others. The
contribution of Leu6 is likely to be essential to the
event. Furthermore, the marked reduction (165-fold) in the
kcat/Km value of
ET
7·His6 for reduced EH and the absence of
cross-linking between Lys288 of ET
7 and
Asp34 of EH (12) may reflect the disordered conformation in
the stabilized C-terminal polypeptide. In this connection, it is worth
noting that two point mutations in the N-terminal region of
human T-protein, H42R (19) and G47R (20), cause the clinical disorder
nonketotic hyperglycinemia. His42 and
Gly47 of the human precursor T-protein correspond to
His10 and Ala15, respectively, of ET.
His42 is conserved in all eight T-proteins reported thus
far, and Gly47 is present in all but E. coli
T-protein (11).
Extensive sequencing of the Lys-C digests of ET·His6
demonstrated 4 sensitive lysine residues, Lys81,
Lys154, Lys288, and Lys360. It is
well known that residues sensitive to proteolytic cleavage are
localized to regions that are solvent-accessible, unstructured, or
flexible. The results are quite reasonable because functional lysine
residues such as Lys81 and Lys288 are situated
in a solvent-accessible location. Lys288 might be masked by
the interaction with Asp34 of EH. The significant
protection by EH of the cleavage at Lys81 indicates a
buried position in the ET-EH complex of the binding site for the second
glutamate residue of folate polyglutamate. In contrast, the promoted
cleavage at Lys360 in the stabilized ET-EH complex suggests
that the region containing Lys360 and probably
Lys352 is located on the surface of the complex. These
results lead us to speculate that the folate binding cavity is formed
by the interaction of ET with EH, in which the binding site for the
second glutamate residue of folate substrate is localized deeper in the cavity than that for the first glutamate residue. Consequently, the
polyglutamate tail of the folate substrate may be inserted into the
bosom of the cavity leaving the pteridine ring near the entrance of the
cavity. This configuration is compatible with the previously revealed
Ordered Ter Bi mechanism of the reverse reaction catalyzed by
T-protein, in which reduced EH is the first substrate that binds ET
followed by folate substrate and ammonia (3). In the study with ET, we
could not distinguish the polypeptide Ala1-Lys360 from the intact polypeptide on
SDS-PAGE nor detect the small peptide that was released,
Ala361-Val-Ala363. However, detection of
polypeptide Ala1-Lys360 in the tryptic digest
of ET in the presence of EH ruled out the possibility that the
positioning of Lys360 near the surface of the ET-EH complex
may be due to the addition of a His6 tag at the C terminus.
The orientation of folate in the cavity is compatible with the
conformation of the lipoyl moiety of H-protein reported by Cohen-Addad
et al. (21). The lipoate arms of pea H-protein in oxidized
or reduced forms, i.e. carrying no methylamine, are located at the surface of the H-protein in a rather flexible conformation, and
following the transfer of methylamine, the cofactor pivots around the
lysine linkage and is locked into the binding cleft of the H-protein.
Therefore, the presence of the pteridine ring harboring a C1 unit
near the surface of the ET-EH complex might facilitate the transfer of
C1 to the lipoyl cofactor of EH.
Recently, Guilhaudis et al. (22) reported an NMR analysis
probing the structural change of pea H-protein bearing two forms of
lipoate cofactor, oxidized and methylamine-loaded, by the addition of
T-protein. They showed that the interaction of both proteins occurs in
a relatively restricted area surrounding the lipoate arm binding site
and Glu42 corresponding to Asp43 of ET.
Consequently, the functionally concentrated area was required on
T-protein involving the H-protein binding site, folate substrate binding site, and other related regions. These results are again consistent with our conclusions. Guilhaudis et al. (22)
proposed that the role of T-protein is not only to locate the
tetrahydrofolate cofactor in a position favorable for a nucleophilic
attack on the methylene carbon but also to destabilize the H-protein in order to facilitate the unlocking of the arm and initiate the reaction.
Its structural nature might be elucidated by crystallographic analysis,
a project that is now in progress.