From the Department of Biochemistry and Conway
Institute of Biomolecular and Biomedical Research, University College
Dublin, Belfield, Dublin 4, Ireland, and § Pfizer Global
Research and Development, Groton, Connecticut 06340
Received for publication, June 19, 2002, and in revised form, October 29, 2002
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ABSTRACT |
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The structural flexibility and thermostability of
glutamate dehydrogenase (GDH) from Clostridium symbiosum
were examined by limited proteolysis using three proteinases with
different specificities, trypsin, chymotrypsin, and endoproteinase
Glu-C. Clostridial GDH resisted proteolysis by any of these enzymes at
25 °C. Above 30 °C, however, GDH became cleavable by
chymotrypsin, apparently at a single site. SDS-PAGE indicated the
formation of one large fragment with a molecular mass of ~44 kDa and
one small one of <10 kDa. Proteolysis was accompanied by the loss of
enzyme activity, which outran peptide cleavage, suggesting a
cooperative conformational change. Proteolysis was prevented by either
of the substrates 2-oxoglutarate or L-glutamate but not by
the coenzymes NAD+ or NADH. Circular dichroism spectroscopy
indicated that the protective effects of these ligands resulted from
fixation of flexible regions of the native structure of the enzyme.
Size-exclusion chromatography and SDS-PAGE studies of
chymotrypsin-treated GDH showed that the enzyme retained its hexameric
structure and all of its proteolytic fragments. However, circular
dichroism spectroscopy and analytical ultracentrifugation showed global
conformational changes affecting the overall compactness of the protein
structure. Chymotrypsin-catalyzed cleavage also diminished the
thermostability of GDH and the cooperativity of the transition between
its native and denatured states. N-terminal amino acid sequencing and
mass spectrometry showed that heat-induced sensitivity to chymotrypsin
emerged in the loop formed by residues 390-393 that lies between
helices Studies of stability in proteins generally encompass their
capacity to retain the native state across wide ranges of temperature, ionic strength, pH, or concentration of denaturant. In the case of
enzymes, there must be a balance between stability and flexibility so
that they combine an ability to survive their environment with the
ability to perform their catalytic function, which frequently requires
rapid reversible conformational changes (1).
Limited proteolysis can be used to probe local structure and
conformational transitions in enzymes. It can also generate fragments that retain certain functional properties of the original enzyme, allowing identification of individual functional domains. This method
in combination with classical protein chemistry methods has been used
widely to define the structural organization of some proteins and to
locate exposed and flexible regions of their native structure
(2-10).
This study examines effects of limited proteolysis at different
temperatures on the structural and catalytic properties of glutamate
dehydrogenase (GDH)1 from
Clostridium symbiosum, a homo-hexameric enzyme for which high resolution crystal structures with and without bound substrate are
available (Protein Data Bank accession numbers 1BGV and 1HRD) (11, 12).
The underlying assumption is that the emergence of proteolytic cleavage
sites in the early stages of unfolding can indicate the relative
flexibility and stability of substructures or domains within the
overall structure. The objective of this study is to identify flexible
or accessible regions in the enzyme structure with a view to the design
of modified forms of clostridial GDH with improved structural
stability. Some of this work has been presented as a poster at a
Biochemical Society Meeting (Galway, Ireland), and a brief
summary was published previously (13).
Materials--
Grade II NAD+ (free acid), NADH
(disodium salt), 2-oxoglutarate (disodium salt), and endoproteinase
Glu-C were obtained from Roche Diagnostics. L-Glutamic
acid, Tris(Tris-[hydroxymethyl]aminomethane), L-1-tosylamido-2-phenylethyl chloromethyl ketone
(TPCK)-treated trypsin, and
1-chloro-3-tosylamido-7-amino-2-heptanone (TLCK)-treated chymotrypsin (the latter two from bovine pancreas) were purchased from
Sigma. Sephadex G-25 (fine), Sepharose CL-6B, and the Superdex 200 HR
10/30 column were from Amersham Biosciences. All other chemicals were
analytical reagent grade.
Enzyme Preparation--
Escherichia coli TG1 pGS516
cells carrying the C. symbiosum GDH gene (14) were grown and
harvested according to the method described earlier (15). Clostridial
GDH was purified by a single step affinity-chromatographic procedure
(16) modified as described elsewhere (17). The enzyme stored in 60%
ammonium sulfate at 4 °C was desalted on a Sephadex G-25 column
before use.
Determination of Protein Concentration--
The enzyme
concentration was determined spectrophotometrically at 280 nm by using
an absorption coefficient of 1.05 liter/g·cm (16).
Enzyme Assay--
GDH activity was measured
spectrophotometrically at 25 °C (Uvikon 941 Plus, Kontron
Instruments S.p.A., Milan, Italy) by recording the change in
A340 resulting from the production of NADH.
Assay solution contained 1 mM NAD+ and 40 mM L-glutamate in 0.1 M potassium
phosphate buffer, pH 7.0, containing also 1 mM EDTA.
Quaternary Structure Analysis--
Size-exclusion chromatography
of GDH samples was conducted on a Amersham Biosciences fast protein
liquid chromatography system using a Superdex 200 HR 10/30 column
equilibrated with 50 mM potassium phosphate buffer, pH 7.0. For calibration of the column, the following molecular mass marker
proteins were used: ferritin (450 kDa), bovine serum albumin (68 kDa),
hen egg albumin (45 kDa), and cytochrome c (12.5 kDa) from
Roche Molecular Biochemicals; SDS-PAGE Analysis--
SDS-PAGE experiments were carried out
according to the Laemmli method (18). GDH samples were prepared with
SDS sample buffer containing 5% Electrospray Mass Spectrometry Measurements--
Electrospray
mass spectrometry was performed on a PE Sciex API 100 single quadrupole
mass analyzer with an electrospray sample interface. Scans were
conducted from 300-2200 in steps of 0.333 atomic mass unit with a
dwell time of 0.75 ms. GDH samples for analysis were first fractionated
on a Poros reverse phase 2H 2.1/30 column using a
water-acetonitrile-trifluoroacetic acid solvent system.
Sedimentation Equilibrium Studies--
Native and
chymotrypsin-treated samples of GDH (both 0.2 mg/ml in 50 mM potassium phosphate buffer, pH 7.0) were run
simultaneously at 50,000 rpm in the Beckman XLI analytical
ultracentrifuge at 4 °C. Interference scans were taken every 30 s after 10-min delay. In total, 120 scans were collected for each
sample. Each data set consisted of radial scans 30-90, and the
time-derivative method was used to analyze the data.
Circular Dichroism Measurements--
CD spectra of native and
chymotrypsin-treated samples of GDH and their thermal denaturation were
recorded in 50 mM potassium phosphate buffer, pH 7.0, using
a Roussel Jouan Dichrographe II instrument. Cell path lengths were 0.1 and 1.0 cm for the measurements in the far- and near-UV ranges of the
spectrum, respectively. Melting curves of the samples (with
concentrations of 0.5 mg/ml) were measured at 220 and 280 nm in the
temperature range of 20-80 °C with a gradient resolution of
0.2 °C. The rate of temperature change was 0.5 °C/min. The
bandwidth of the equipment was 0.5 nm with a response time of 4 s.
Amino Acid Sequence Analysis--
The samples of GDH treated by
proteinases were subjected to SDS-polyacrylamide gel electrophoresis.
Gels were electroblotted onto Sequi-Blot polyvinylidene difluoride
membrane (Bio-Rad), and proteolytic fragments were detected by staining
with Coomassie Brilliant Blue. Bands were subjected to sequence
analysis by automated Edman degradation on an Applied Biosystems
Procise 494 protein sequencer.
Proteolytic Cleavage and Protection by Substrates--
When native
clostridial GDH (1 mg/ml) was incubated with trypsin or chymotrypsin
(0.1 mg/ml) at 25 °C in 100 mM potassium phosphate, pH
7.0, there was practically no effect on either GDH activity or the
structural integrity of the enzyme as measured by SDS-PAGE (Fig.
1, lanes a1 and
b1). Activities after 30-min incubation with trypsin and
chymotrypsin were 100 and 95%, respectively. An increase in the
incubation temperature to 50 °C, however, resulted in rapid
inactivation of GDH in the presence of chymotrypsin (residual GDH
activity after 30 min was <4%), although virtually full enzyme activity (~98%) remained after similar treatment with trypsin. SDS-PAGE showed no changes in the structure of GDH after trypsin treatment (Fig. 1, lane a2), but during incubation with
chymotrypsin, a large intermediate fragment of ~45 kDa was rapidly
further degraded into fragments too small for detection on the gel
(Fig. 1, lane b2).
Chymotryptic cleavage of native GDH could be largely prevented by the
addition of the substrate 2-oxoglutarate (Fig.
2). This protective effect was enhanced
by the coenzyme NAD+, presumably by forming the
catalytically non-productive complex (enzyme-NAD+-2-oxoglutarate), even though NAD+
on its own had little effect (Fig. 2). Similar results were obtained with L-glutamate and NADH singly and in combination (data
not shown). Corresponding to the structural protection, in these
experiments there was also retention of catalytic activity.
To retard the secondary cleavage of the proteolytic fragments, the
proteolysis by chymotrypsin was carried out at intermediate temperatures. The SDS-PAGE pattern of GDH fragments obtained by chymotrypsin treatment at 30, 35, and 40 °C shows (Fig.
3) that proteolysis could be achieved
even at 30 °C, although the rate of digestion depended strongly on
the incubation temperature. For incubation temperatures of 30, 35, and
40 °C, residual GDH activities after 20 and 60 min of incubation
were as follows: at 30 °C, 85 and 40%; at 35 °C, 38 and 22%;
and at 40 °C, 15 and 1%. Almost complete fragmentation of the
native subunit was obtained at 40 °C over 60 min of incubation.
Three major bands could be seen on the SDS gel: (a) a band
in the native subunit position (50 kDa); (b) a large
fragment ~10% smaller than the native subunit; and (c) a
band near the bottom of the gel corresponding to a molecular mass of
<10 kDa. In contrast to the situation at 50 °C, proteolytic fragments obtained at lower incubation temperatures appeared to be more
stable. SDS-PAGE experiments on 12% SDS gels with the use of smaller
amounts of protein to give sharp bands provided an estimate of 44 kDa
for the size of the large GDH fragment.
Influence of Buffer Composition--
Inactivation of bovine liver
GDH by trypsin is much faster in Tris buffer than in phosphate buffer
(19), confirming the view that these two buffers have strikingly
different effects on the structure and functional properties of this
enzyme. In search of possible similar effects, the proteolysis of
clostridial GDH by chymotrypsin was carried out in both 50 and 100 mM Tris or potassium phosphate buffer, pH 7.0. At higher
concentrations of buffers, clostridial GDH showed better resistance
against chymotrypsin digestion (data not shown). Also, in the potassium
phosphate buffer, clostridial enzyme and its proteolytic fragments were
more stable than they were in the Tris buffer. However, the presence of
150 mM NaCl in the incubation mixture dramatically
increased the stability of clostridial GDH against chymotryptic
digestion in either one of these buffers (data not shown).
Circular Dichroism Studies--
Since clostridial GDH was
accessible to digestion by chymotrypsin at elevated temperatures but
not at 25 °C and in view of the specificity of chymotrypsin for
cleavage at aromatic amino acid side chains it seemed that the near-UV
region of the CD spectrum of clostridial GDH might reveal relevant
temperature-dependent conformational changes. In fact, CD
spectra recorded between 35 and 50 °C (Fig.
4) showed 50% increase in CD signal in
the near-UV spectra under conditions where the clostridial enzyme
remained completely active in the absence of proteolysis. Thus, above
35 °C, clostridial GDH undergoes conformational changes that alter the environment of aromatic residues. This alteration together with increased flexibility would account for the acquired
susceptibility to chymotryptic digestion.
An attempt was made to correlate the protective effects of specific
ligands and NaCl with the changes in enzyme conformation observed by
CD. Thermal denaturation curves of clostridial enzyme were measured by
CD (near-UV region, 280 nm) in the presence of 50 mM
2-oxoglutarate or L-glutamate or 150 mM NaCl
(data not shown). In the absence of these ligands, a gradual increase
of the CD signal was seen above 35-40 °C (as in Fig. 4). However,
in the presence of NaCl, the CD signal stayed unchanged up to 50 °C. Moreover, in the presence of 2-oxoglutarate or L-glutamate,
conformational changes were detected only above 55 °C. The effects
of NAD+ and NADH with or without 2-oxoglutarate and
L-glutamate could not be measured by this method because of
the high absorbance of these coenzymes. Nevertheless, these results
suggest that the protection by specific ligands 2-oxoglutarate and
L-glutamate as well as NaCl against chymotryptic digestion
results from immobilization of flexible regions of the native structure.
Effect of the Chymotrypsin Treatment on Enzyme Quaternary
Structure--
Native clostridial GDH exists as a homo-hexamer
composed of identical subunits (11, 20) with a molecular mass of 49,165 Da predicted from the gene sequence (14). To discover the effect of
chymotryptic digestion on the hexameric structure, 1 mg/ml native
clostridial GDH in 50 mM potassium phosphate, pH 7.0, was incubated at 40 °C with chymotrypsin (final concentration, 0.1 mg/ml). Aliquots withdrawn after 0-, 15-, 30-, and 45-min incubation were cooled on ice. Proteolysis was stopped immediately by the addition
of phenylmethylsulfonyl fluoride in 2500-fold molar excess relative to
the proteinase. Samples were prepared for analysis by size-exclusion
chromatography and SDS-PAGE, and their specific activities were also
measured. The results summarized in Table I showed that after 15 min >50% GDH
subunits were already digested and only 16% of the subunits remained
uncleaved after 45 min of incubation. However, the most striking
conclusion from the results of Table I was that the loss of GDH
activity proceeded faster than proteolytic cleavage. For example, the
residual GDH activity after 15-min incubation was 23%, although only
half of the enzyme subunits were cleaved. This possibly implies that
cleavage leads to cooperative conformational responses in neighboring
uncleaved subunits.
In size-exclusion chromatography for all three proteolysed samples, a
small amount of material increasing with the time of proteolysis eluted
from the Superdex 200 HR 10/30 column in a position corresponding to a
molecular size much smaller than that of the clostridial GDH monomer.
However, in all three cases, the great majority of protein material
eluted in exactly the same position as native hexameric enzyme. For
example, in sample 3 (Table I), almost 80% of the enzyme retained its
hexameric structure, although >95% of the original activity was lost.
SDS-PAGE of these hexameric fractions (Fig.
5) showed that they contained both major fragments of the digested enzyme. In this connection, it is important to note that retention of hexameric structure and of all fragments is
not the result of internal disulfide linkage among these fragments or
subunits. Clostridial GDH contains only two cysteine residues (14) in
each monomer (Cys-144 and Cys-320) which are not involved in either
intrasubunit or intersubunit disulfide linkage in the native hexamer
(11). Moreover, it is clear from the sequence (Fig.
6) that any single peptide fragment
cleaved from either the N or C terminus of the clostridial enzyme must
have a molecular size of at least 15 kDa to be able to accommodate one
of these cysteine residues.
N-terminal Amino Acid Sequence Analysis--
The N-terminal amino
acid sequence was determined for all three bands from lane 3 of the SDS gel (Fig. 5). The two upper bands had
an identical N-terminal sequence (SKYVDRVIAE ... ), which is the
N-terminal sequence of native GDH (Fig. 6) (14), indicating that the
N-terminal region of the clostridial enzyme is quite resistant to
attack by chymotrypsin. It appears that chymotrypsin cleaves a peptide
bond ~50 residues in from the C terminus of the native protein. The
N-terminal sequence of the lower band was identified as
TAEEVDSKLH ... , indicating cleavage of the peptide bond between
residues Trp-393 and Thr-394 in the GDH sequence (Fig. 6).
Mass Spectrometry--
LC-MS was used to determine the molecular
masses of the chymotryptic fragments of clostridial GDH. Hexamer
fractions of samples 1 and 4 (samples of GDH after 0- and 45-min
incubation with chymotrypsin, respectively, Table I) eluted from the
Superdex 200 column were loaded separately onto a reverse phase column
attached to a mass spectrometer. Undigested GDH (sample 1) eluted from
the column as a single peak with a retention time of 77.6 min (data not
shown). Mass analysis of this peak gave a value of 49,173 Da
within 0.02% of the theoretical mass of the native subunit of
clostridial GDH (theoretical mass 49,165 Da). In contrast, sample 4 gave two main peaks (Fig. 7A).
The first small peak eluted with a retention time of 68.0 min and
clearly showed a molecular mass of 5,907 Da (Fig. 7B). This
corresponds closely to the mass of peptide fragment-(394-449)
of clostridial GDH (theoretical mass 5,908 Da). The second larger peak
eluted with a retention time of 76.8 min and had a back shoulder,
suggesting the presence of multiple species. The estimated molecular
mass of the major component (Fig. 7C, 43278 Da),
was within 0.01% of the theoretical mass of the peptide
fragment-(1-393) of clostridial enzyme (theoretical mass 43,275 Da).
Minor components with molecular masses of 43,007, 43,513, and 49,178 Da
can also be seen in Fig. 7C. The mass of 49,178 Da
corresponds to the undigested subunit. In fact, an analysis of the back
shoulder of the second peak showed a higher proportion of material with
a molecular mass of 49,178 Da (data not shown), indicating the presence
of undigested subunits in this peak. The nature of the component with
the mass of 43,513 Da is not obvious, but the mass of 43,007 Da could
be correlated with the predicted mass (43,002 Da) of the
fragment-(1-391) of the clostridial enzyme. Attempts to find a
fragment with a molecular mass of 6182 Da (corresponding to the
residual peptide fragment-(392-449)) failed. This was anticipated because of the good accessibility of the peptide bond between Trp-393
and Thr-394, resulting in the initial formation of the peptide-(394-449) with a mass of 5908 Da. However, as shown later, after chymotrypsin treatment of clostridial GDH, the C-terminal fragment of the peptide-(1-393) becomes very flexible and it could be
accessible for further attack by chymotrypsin as well as by other
proteases. In this particular case, chymotrypsin probably attacked the
peptide bond between Leu-391 and Ser-392 of the peptide fragment-(1-393) with the release of a dipeptide and formation of the
fragment-(1-391).
Physical Properties of GDH after Chymotryptic Cleavage--
As
seen above, chymotrypsin cleaved clostridial GDH mainly between Trp-393
and Thr-394 with the formation of two polypeptide fragments-(1-393 and
394-449). However, the digested enzyme retained its hexameric
structure and contained both of these fragments. Properties
of this nicked enzyme were investigated by CD and analytical ultracentrifugation. As a control, the native enzyme was analyzed under
the same conditions. Fig. 8 shows CD
spectra measured at 20 °C for native and chymotrypsin-treated
samples of clostridial GDH in the near- (A) and far-
(B) UV regions. It is clear that the chymotrypsin treatment
affected the secondary structure of clostridial GDH. In the near-UV
(Fig. 8A), the CD spectrum of the treated enzyme showed an
elevated ellipticity reflecting changes in the environment of aromatic
side chains. Such changes in the near-UV CD spectra were anticipated
from the experimental results discussed above. However,
chymotrypsin-treated enzyme showed a decreased CD signal in the far-UV
region (Fig. 8B), indicating more widespread changes in the
secondary structure. Analytical ultracentrifugation studies yielded
sedimentation coefficients of 13.2 and 11.7 S, respectively, for the
native and chymotrypsin-treated enzymes (data not shown). These results
point to an increase in relative hydrodynamic radius of the proteolysed
samples and thus also to substantial changes in the compactness of the
digested enzyme.
To find out the effect of chymotrypsin digestion and
consequent conformational changes on the thermal stability of
clostridial GDH, the CD signals at 220 nm were monitored continuously
with increasing temperature. This experiment showed (Fig.
9) a melting point of
chymotrypsin-digested GDH of 56.5 °C in comparison with 62.2 °C
for the native enzyme, indicating a significant decrease in structural
stability of the protein after cleavage. Also, denaturation of the
chymotrypsin-treated enzyme occurred over a wider temperature range,
suggesting a less cooperative transition from the folded to the
unfolded state than in the native enzyme.
Further Digestion of Chymotrypsin-treated GDH with Other
Proteinases--
As outlined above, the treatment of clostridial GDH
with chymotrypsin produces major conformational changes affecting both stability and cooperativity. The most flexible region of the enzyme appears to be the loop lying between helices
A further attempt was made to estimate the extent of this most flexible
section of the clostridial GDH molecule. Fig. 6 shows that several
glutamic acid residues are located around the chymotryptic and tryptic
digestion sites of clostridial GDH. Specifically, Glu-389 is located at
the end of helix The results described here show parallels in some respects with
earlier studies on limited proteolysis of glutamate dehydrogenase from
bovine liver (19, 21-25). Chymotrypsin treatment produced a cleavage
product with a molecular size ~5000 Da smaller than the native enzyme
(according to SDS-PAGE) but resulted in severalfold activation (22,
24). This intermediate had reportedly lost the ability to respond to
allosteric activation by ADP (22), although the extent and direction of
regulatory changes depended on the pH and composition of the assay
mixture (23, 25). As in the present case, the chymotryptic cleavage
produced no change in the quaternary structure or molecular weight of
the enzyme. Also, the bovine liver enzyme was very resistant to
trypsin, but the single chymotryptic cleavage opened up the enzyme to
further attack and inactivation by trypsin.
In the case of the bovine enzyme, it is not yet clear at which end of
the polypeptide chain the cleavage occurs, but in clostridial GDH, we
are now able to locate the site precisely not only in the sequence but
also in the three-dimensional structure. The loop in question comes
between two long helices, Native clostridial enzyme exists as a homo-hexamer with a 32-structural
symmetry (11, 20) and, as shown above, retains its hexameric structure
even after treatment with chymotrypsin. Fig.
11 represents the crystal structure of
the hexamer of the clostridial enzyme and shows locations of these
thermally sensitive loops, which are in a large cavity round the 3-fold
axis of the hexamer. Thus, there are two such cavities, one for each
trimer (each containing three sensitive loops), on either side of the GDH molecule. It is obvious from the structure of GDH (Fig. 11) that
the molecule of chymotrypsin would not readily be able to approach the
cleavage site because it is too large to penetrate deep into the
cavity. This must imply that as the temperature is raised, the
sensitive loop becomes in some way more accessible. One possibility
would be that there is a transient dissociation to monomers, which
would then make the loop fully accessible. If so, however, it would be
difficult to explain the failure to observe cleavage by chymotrypsin at
a number of other potential sites such as those in the loop joining
15 and
16 in the folded structure of the enzyme.
INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
EXPERIMENTAL PROCEDURES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
-amylase (200 kDa), carbonic anhydrase
(29 kDa), and aprotinin (6.5 kDa) from Sigma. The elution flow rate was
0.5 ml/min.
-mercaptoethanol as a reducing
agent. The gels were stained with Coomassie Brilliant Blue R-250, and
protein bands were analyzed with the GDS8000 Gel Documentation
System (UVP Inc.) using GelWorks 1D Intermediate software (Non-Linear Dynamics Ltd.).
RESULTS
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
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Fig. 1.
12% SDS-PAGE of clostridial GDH treated with
trypsin and chymotrypsin. Samples of GDH were incubated
over 30 min with trypsin (a) and chymotrypsin (b)
at 25 °C (lanes 1) and 50 °C
(lanes 2) in 100 mM potassium
phosphate, pH 7.0. Untreated GDH was used as a control (lane
0). Positions of molecular mass marker proteins are given on
the left.
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Fig. 2.
12% SDS-PAGE of clostridial GDH treated with
chymotrypsin in the presence of specific ligands. Samples of GDH
(1 mg/ml) were incubated with chymotrypsin (0.1 mg/ml) at 50 °C in
the absence and the presence of 1 mM NAD+ or 50 mM 2-oxoglutarate or both together. Aliquots were withdrawn
after 10 (lanes 1), 30 (lanes 2), 60 (lanes
3), and 120 (lanes 4) min of incubation, and
proteolysis was stopped by the addition of phenylmethylsulfonyl
fluoride (2500-fold molar excess relative to the proteinase). A sample
of GDH without chymotrypsin treatment was prepared for the gel
similarly and used as a control (lanes 0).
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Fig. 3.
12% SDS-PAGE of clostridial GDH treated with
chymotrypsin at different incubation temperature. Samples of GDH
were incubated with chymotrypsin over 20 min (lanes 1) and
60 min (lanes 2) at 30, 35, and 40 °C as indicated.
Untreated GDH was used as a control (lane 0). Positions of
molecular mass marker proteins are given on the left.
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Fig. 4.
Near-UV circular dichroism spectra of native
clostridial GDH measured at different temperatures as indicated.
Concentration of GDH was 0.5 mg/ml in 50 mM potassium
phosphate buffer, pH 7.0. CD spectra were measured in a quartz cell
with path length of 1 cm.
Some characteristics of chymotrypsin-treated samples of clostridial
GDH
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Fig. 5.
12% SDS-PAGE of clostridial GDH digested
with chymotrypsin before (lane 2) and after
(lane 3) size-exclusion chromatography (hexameric
fraction). The sample of GDH (1 mg/ml) was incubated with
chymotrypsin (0.1 mg/ml) over 30 min at 40 °C in 50 mM
potassium phosphate buffer, pH 7.0. Untreated GDH was used as a control
(lane 1). Major peptide fragments of the
chymotrypsin-treated GDH are indicated on the right and were
used for N-terminal amino acid sequencing.
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Fig. 6.
Amino acid sequence of clostridial GDH with
the secondary structural elements. Useful Protein Data Bank
accession numbers for clostridial GDH are 1BGV and 1HRD. The sizes of
helices (rectangles),
sheets (arrows),
and connecting loops (lines) correspond to the number of
amino acids involved in these structural elements. The position of the
major chymotrypsin digestion site is shown by the large vertical
arrow. Secondary proteolytic digestion sites are shown by
small vertical arrow.
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Fig. 7.
LC-MS analysis of the chymotrypsin-treated
sample of clostridial GDH. The sample loaded onto LC-MS was the
hexameric fraction of the chymotrypsin-treated GDH (Table I, sample 4).
A, elution profile of the sample. B and
C, deconvoluted mass spectra from peaks 1 and 2, respectively.
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Fig. 8.
CD spectra for the native
(1) and chymotrypsin-treated (2)
samples of clostridial GDH at near-UV (A) and far-UV
(B) regions. All spectra were measured at
20 °C and normalized to the protein concentration. Path length of
the cells for CD measurements was 1 cm for the near-UV and 0.1 cm for
the far-UV regions. In these experiments, the hexameric fractions of
the untreated and chymotrypsin-treated samples of GDH (Table I, samples
1 and 4) were used.
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Fig. 9.
Thermal denaturation of the native
(1) and chymotrypsin treated (2)
samples of clostridial GDH measured in the far-UV region of CD spectra
(220 nm). Concentration of both samples of GDH was 0.1 mg/ml in 50 mM potassium phosphate buffer, pH 7.0. The hexameric
fraction of chymotrypsin-treated samples of GDH (Table I, sample 4) was
used in the experiment.
15 and
16 of the enzyme (Figs. 6 and
10), as this was the first point where
proteolysis occurred as the temperature was raised. As discussed above,
apart from the residue Trp-393, chymotrypsin attacks also the peptide bond between Leu-391 and Ser-392, although much more slowly. However, it was not clear whether this minor cleavage at the position Leu-391 required prior cleavage at Trp-393. Even though the fragment-(392-449) was not seen, this could merely reflect rapid secondary cleavage at
Trp-393. However, taking into consideration that one of the potential
tryptic digestion sites (Arg-390) lies just beside Leu-391 and the fact
that native clostridial GDH is quite resistant to trypsin digestion
(even at 50 °C), it seems probable that the second chymotryptic site
became available only after digestion at Trp-393. To test
this view, the hexameric fraction of the chymotrypsin-treated enzyme
was incubated over 45 min with trypsin at 25 °C. SDS-PAGE of the
incubation mixture showed virtually no difference between samples
before and after incubation with trypsin (data not shown). However,
LC-MS analysis of the same incubation mixture clearly revealed the
following two GDH fragments: 1) 5910 Da, which is the same 394-449
chymotryptic fragment of the enzyme, and 2) 42,895 Da, which
corresponds within 0.01% with the mass of the tryptic fragment-(1-390) of the clostridial enzyme (theoretical mass 42,889 Da). These results first of all indicate that both chymotryptic fragments of clostridial enzyme (1-393 and 394-449) retain their compact secondary structure and remain resistant to trypsin with the
exception of a small segment (tripeptide) at the C terminus of 1-394,
which becomes exposed and accessible to digestion. Secondly, they
support the above assumption that the second chymotrypsin digestion
site in clostridial enzyme (Leu-391) is a result of conformational
changes in the loop area between helices
15 and
16 of the GDH structure caused by the first cleavage at
Trp-394.
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Fig. 10.
Ribbon diagram of the clostridial
GDH subunit (Protein Data Bank accession number 1HRD). The
C-terminal fragment-(394-449) (Fig. 6) consisting of two helices,
16 and
17, is shown in yellow.
The helix
15 and the loop connecting with helix
16 are shown in green. Locations of the
primary chymotryptic (shown in red) and tryptic digestion
sites are indicated.
15 and just beside the tryptic cleavage
site (Arg-390), and two others, Glu-396 and Glu-397, at the beginning
of helix
16 are just two residues away from the main
chymotryptic digestion site Trp-393. However, incubation of
chymotrypsin-treated enzyme with endoproteinase Glu-C had no effect on
the molecular masses of either of the polypeptide fragments, 1-393 or
394-449. This finding indicates that the proteolysis by chymotrypsin
does not expose any new sites for cleavage by Glu-C and that over the
temperature range studied only the tripeptide loop-(391-393) (Figs. 6
and 10) becomes susceptible to proteolysis.
DISCUSSION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
15 and
16, highlighted in Fig. 10.
16 and
17 (e.g. Tyr-420, Tyr-424). Also, trypsin was found only to cleave at Arg-390 after the
chymotryptic cleavage at Trp-393, whereas in a monomer, not only this
site but also a number of others might be readily accessible even
without prior proteolysis by chymotrypsin.
View larger version (78K):
[in a new window]
Fig. 11.
A wire-frame structure of the clostridial
GDH hexamer with ribbon representation of locations of the flexible
region ( 15 and
16) in each subunit.
Different colors were used for different subunits. The
primary chymotryptic digestion site (Trp-393) is highlighted in
gray for all six subunits. The structure of bovine
pancreatic
-chymotrypsin (Protein Data Bank accession number 5CHA)
is shown in magenta for comparison. The figure was created
using RasMol version 2.7 by Herbert J. Bernstein (based on RasMol 2.6 by Roger Sayle).
Therefore, it is difficult to escape the conclusion that the increased
temperature leads to a specific conformational arrangement that makes
the target loop available to chymotrypsin. Inspection of the structure
shows that the two helices, 15 and
16,
which are linked by the sensitive loop, are not directly involved in the formation of the intersubunit contacts, and in principle, such a
conformational rearrangement might happen by a large movement of these
two helices,
15 and
16, outward from the
3-fold axis, projecting the loop into a much more exposed position.
In connection with this loop, it is interesting to note that the major
structural difference between bacterial and mammalian GDHs is the large
48-amino acid insertion between 15 and
16 helices and starting right after residue Trp-393 (14). The recently determined crystal structure of the bovine GDH in a complex with NADH,
glutamate and GTP have revealed that this 48-residue insertion forms an
"antenna" structure lying immediately adjacent to the 3-fold axis
of the hexamer, and it has been suggested that this domain of the
mammalian enzyme is involved in the regulation by allosteric effectors
GTP and ADP as well as by coenzymes NAD+ and NADH (26, 27).
It was also suggested that allosteric effects of these regulators on
enzyme activity (activation by ADP and inhibition by GTP) are realized
via conformational changes in this regulatory domain affecting the
energy required for the enzyme to open and close the active center
cleft during the catalytic cycle. On the other hand, these
allosteric regulators GTP and ADP have opposite effects not only on the
catalytic activity but also on the ability of this enzyme to associate
into high molecular weight aggregates by polymerization of hexamers
along the 3-fold axis of the enzyme (28, 29). It is reasonable to
surmise that the realization of such effects by GTP and ADP requires
substantial conformational changes in the antenna domain, which is
involved in the contact area among hexamers. Therefore, the two helices of the bovine enzyme flanking this antenna domain (corresponding to the
clostridial helices
15 and
16) must have
enough flexibility to allow these conformational changes. Clearly, one
cannot reliably extrapolate from the complex bovine GDH structure to
the simpler clostridial structure. Nevertheless, the helices in
question are a conserved feature, and since the clostridial GDH also
displays allosteric behavior (15, 30, 31), it is not inconceivable that
these flexible helices also play a key role in subunit interaction in
the bacterial enzyme.
An interesting question in relation to the catalytic
mechanism of clostridial GDH is the role of glutamate. Crystallography has suggested that the amino acid substrate triggers a closure of the
cleft separating the two domains of the monomer to enclose the active
site of the enzyme (12). A comparison of the structures for the free
clostridial enzyme (open) and for the enzyme-glutamate complex (closed)
reveals that this hinge closure causes remarkably little change in the
location of the sensitive loop. Hinge closure alone cannot therefore
explain the pronounced protection by glutamate against proteolysis by
chymotrypsin. However, the structure of the enzyme-glutamate complex
shows that the carboxylate groups of the bound substrate interact
directly with Ser-380 and indirectly via Lys-113 with Asn-373 (12).
Both of these residues are located in the helix 15, and
it seems reasonable to suggest that the complexation with glutamate may
impede any tendency for
15 and
16 to
swing outward as suggested above, thus protecting the enzyme from
chymotrypsin digestion.
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ACKNOWLEDGEMENTS |
---|
We thank Dr. J. G. Stroh (Pfizer Global Research and Development, Groton, CT) for access to the mass spectrometer and useful discussions, Michele H. Rosner for analytical ultracentrifugation studies, and Anthony J. Lanzetti for protein sequence analysis. We are grateful to the members of the Sheffield University crystallography group (Sheffield, United Kingdom) including Prof. D. W. Rice and Drs. P. J. Baker, T. J. Stillman, and K. L. Britton for providing the coordinates of the C. symbiosum GDH hexamer.
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FOOTNOTES |
---|
* This work was supported in part by a grant from Pfizer Inc.The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
¶ To whom correspondence should be addressed. Tel.: 353-1-716-1547; Fax: 353-1-283-7211; E-mail: paul.engel@ucd.ie.
Published, JBC Papers in Press, October 31, 2002, DOI 10.1074/jbc.M206099200
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ABBREVIATIONS |
---|
The abbreviations used are: GDH, glutamate dehydrogenase (EC 1.4.1.2); LC-MS, liquid chromatography-mass spectrometry.
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