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INTRODUCTION |
Heparin-like glycosaminoglycans, such as heparin and heparan
sulfate, are acidic polysaccharides that play a role in many central
biological processes, such as cell proliferation and signaling (1, 2).
However, attempts to determine whether heparin-like glycosaminoglycans
are involved in a particular biological process have been hampered by
the lack of tools available to study these substrates.
One such tool, under development in our laboratory, is the heparinases,
bacterially derived lyases. Heparinase I from Flavobacterium heparinum is a 43-kDa enzyme that cleaves primarily
heparin-like regions of heparin-like glycosaminoglycans
(i.e. regions containing a high degree of sulfation with
primarily iduronic acid as the uronic acid component) (3, 4).
Heparinase I has been used in a variety of circumstances to highlight
the importance of heparin-like glycosaminoglycans in such diverse
biological processes as angiogenesis (5) and development (6).
To extend the capabilities of the heparinases, we have undertaken a
series of biochemical studies aimed at identifying important functional
residues as well as elucidating the enzyme's mode of action. Through a
combination of chemical modification, proteolytic mapping, and
site-directed mutagenesis, we have identified Cys135 (7),
His203 (8), and Lys199 (9) as amino acid
residues important for heparinase I activity. Furthermore, using a
matrix-assisted laser desorption ionization/mass spectrometry and
capillary electrophoresis methodology, we discovered that the mode of
action of heparinase I is primarily exolytic and processive,
i.e. the enzyme preferentially cleaves first at the
nonreducing end and then proceeds to cleave the substrate sequentially
without releasing it (10).
Previously, it has been noted that calcium is required for full
heparinase I activity (11). One possible explanation for this observed
increase is that heparinase I binds calcium and that this interaction
in some way facilitates cleavage of the polymer substrate. However, it
is also known that calcium interacts in a highly specific way with
heparin, inducing a conformational change in the polymer chain (12,
13). Thus, another possibility is that heparinase I can act only on the
calcium-induced conformation of heparin. If this were the case, then
the enzymatic activity of heparinase I could be affected by increasing
calcium concentration without any direct interaction between calcium
and the enzyme.
In an effort to further understand the role of calcium in heparinase I
activity, we undertook a series of experiments to determine whether
heparinase I binds calcium and, if so, at what sites and whether this
interaction is important for the proper functioning of the enzyme. We
sought to study the interaction and stoichiometry of calcium binding to
heparinase I by a combination of fluorescence spectroscopy, chemical
modification, and tryptic mapping.
In this study, we find that calcium indeed binds heparinase I. Furthermore, through a combination of chemical modification and peptide
mapping studies, we identify two calcium-coordinating motifs, with
either one or both of these sites being important for the critical
calcium-heparinase I interaction. In the accompanying study (14), we
use the information derived herein to design a series of site-directed
mutants of the calcium-binding sites of heparinase I to identify
particular amino acids involved in calcium binding and critical for
proper enzymatic functioning.
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EXPERIMENTAL PROCEDURES |
Chemicals and Materials--
Urea, Tris, and trifluoroacetic
acid were from J. T. Baker Inc. Dithiothreitol, EDTA, and
MOPS1 were obtained from
Sigma. Bovine serum albumin and Chelex resin were purchased from
Bio-Rad. TbCl3, LuCl3, and CaCl2 as
well as the chemical modification reagents
N-ethyl-5-phenylisoxazolium-3'-sulfonate (Woodward's
reagent K (WRK)), 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride (EDAC), and glycine methyl ester were all purchased from
Aldrich. EDAC was used as received. WRK was recrystallized prior to
use. Trypsin was obtained from Boehringer Mannheim. Heparin (from
porcine intestinal mucosa with an average molecular mass of 13 kDa) was
obtained from Celsus Laboratories (Cincinnati, OH).
Heparinase I Activity Assay--
Native heparinase I from
F. heparinum was purified as described previously (15, 16).
The UV 232 nm assay to quantify heparinase I enzymatic activity was
similar to that reported elsewhere (17). Briefly, the course of the
reaction was monitored by measuring the increase in absorbance at 232 nm from the
4,5 bond of the product of heparinase
cleavage as a function of time under saturating substrate
concentrations. With heparin as the substrate, the reaction was carried
out at a concentration of 4 mg/ml in 100 mM MOPS and 5 mM calcium acetate, pH 7.0. The temperature for all
enzymatic activity measurements was kept constant at 30 °C. For the
inactivation kinetic profiles, activity was measured as outlined above
at precise time points after addition of the modifying reagent.
Terbium Titrations of Heparinase I--
The titrations of
heparinase I with terbium were completed by adding aliquots of a
terbium stock solution (in 10 mM MOPS and 0.1 M
KCl, pH 6.5) to a solution containing heparinase I (4.6 µM). To maintain a constant protein concentration, the
same amount of heparinase I (4.6 µM) was present in the
terbium stock solution as was present in the cuvette. The concentration
of the terbium solution was determined by EDTA titration in the
presence of a xylenol orange indicator (18). To ensure accurate
readings, all solutions, except the terbium stock solution, were run
through a chelating column (Chelex resin) to remove trace contaminants. After addition of a terbium aliquot, the sample was mixed and allowed
to come to equilibrium for 15 min. Fluorescence measurements were
recorded on a FluoroMax fluorescence spectrometer (Spex Industries, Edison, NJ). The geometry of fluorescence detection was 90°. All measurements were recorded using a quartz cell (Starna Cells) with a
1.0-cm path length, and the sample temperature was maintained at
25 °C using a circulating water bath. The excitation wavelength was
either 488 nm (direct excitation of terbium) or 280 nm (excitation of
nearby tyrosine residues); the emission wavelength was 545 nm.
For the calcium competition titrations, to a solution of heparinase I
(4.6 µM) plus 8 molar eq of terbium were added aliquots of a 50 mM calcium solution that also contained 4.6 µM heparinase I. After each addition, the solution was
thoroughly mixed and allowed to stand for 15 min before a measurement
was taken. In none of the experiments was protein precipitation evident.
Effect of Lanthanides on Heparinase I Activity--
To determine
the effect of Tb3+ and Lu3+ on heparinase I
activity, heparinase I was preincubated for 15 min with increasing
amounts of a solution of 10 mM MOPS and 0.1 M
KCl, pH 6.5, with either Tb3+ and Lu3+. At this
point, the activity of the heparinase I solution was measured using the
232 nm assay. The substrate solution was 4 mg/ml heparin and 5 mM calcium in 10 mM MOPS and 0.1 M
KCl, pH 6.5. The concentrations of the lanthanide stock solutions were determined as outlined above. Control reactions were run in the absence
of lanthanide.
Formation and Degradation of the Ketoketenimine Intermediate from
WRK--
Upon addition of WRK to an enzyme solution, the actual agent
that modifies nucleophilic amino acids is not WRK itself; rather WRK is
converted into a reactive intermediate that binds to selective amino
acids in a protein (19). Therefore, to accurately model the kinetics of
WRK modification of heparinase I, it is necessary to know the
concentration of this intermediate, the ketoketenimine, as a function
of time. The concentration of the ketoketenimine can be determined by
monitoring an aqueous solution of WRK at 340 nm, where the
ketoketenimine is the only species of WRK that absorbs appreciably (
= 4730 cm
1 M
1) (19, 20). At pH
7.0, the conversion of 50 µM WRK to the ketoketenimine
and its subsequent degradation were determined in 100 mM
MOPS by monitoring the change in absorbance at 340 nm every 30 s
for 10 min. Stock solutions of WRK were made fresh with 0.1 M HCl at 4 °C. A cuvette containing an equivalent amount of 0.1 M HCl instead of WRK was used as a blank. To
determine the rate constants of formation (k") and
degradation (k') of the ketoketenimine, Equation 1 was
used.
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(Eq. 1)
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In this equation, t is the measured time,
[I] is the concentration of the ketoketenimine
intermediate, and [W]0 is the initial concentration of
WRK (50 µM) (19).
Inactivation of Heparinase I with WRK--
Heparinase I (30 µg/ml) was inactivated with 0.1-0.4 mM WRK at room
temperature. The control mixture contained no WRK, but an equivalent
amount of 0.1 M HCl. Reactions were carried out in 100 mM MOPS, pH 7.0. At fixed time intervals, aliquots were withdrawn for the UV 232 nm activity assay. The kinetics of WRK inactivation of heparinase I were determined by plotting the natural log of percent activity versus an adjusted time term (to
account for the formation and decomposition of the ketoketenimine
intermediate). This adjusted time term was calculated according to
Equation 2.
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(Eq. 2)
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Ca2+ Protection of WRK Inactivation of Heparinase
I--
To investigate the ability of Ca2+ to protect the
enzyme against modification by WRK, heparinase I (30 µg/ml) was first
incubated with different concentrations of Ca2+ (ranging
from 100 µM to 20 mM) for 30 min at pH 7 before 50 µM WRK was added to the reaction mixtures. The
time course of inactivation was then determined. An activity assay was
also performed with a control mixture with no prior addition of
Ca2+.
Tryptic Digest and Protein Sequence Analysis--
Tryptic
digests of heparinase I were performed essentially as described
previously (7). To 16 µg of heparinase I was added 4 mM
WRK; the sample was allowed to incubate for 30 min at room temperature.
A 10-fold excess of glycine methyl ester was added to quench the
reaction. The enzyme was then denatured in 50 µl of 8 M
urea and 0.4 M ammonium carbonate, reduced with 5 mM dithiothreitol at 65 °C, cooled to room temperature,
and alkylated with 10 mM iodoacetamide for 15 min. The
reaction was quenched with water by bringing the total reaction volume
to 200 µl. To the above reaction was added 4% (w/w) trypsin, and the
digestion was carried out at 37 °C for 24 h. The proteolytic
reaction was terminated by freezing at
20 °C. The digest was
separated using gradient reverse-phase HPLC (2-80% acetonitrile in
0.1% trifluoroacetic acid for 120 min). Tryptic peptides were
monitored at 210, 277, and 320 nm and collected. Based on the peptide
peaks monitored at 320 nm, five peaks were collected and sequenced
using an on-line Model 120 phenylthiohydantoin-derivative analyzer
(Biopolymers Laboratory, Center for Cancer Research, Massachusetts
Institute of Technology). To determine whether preincubation with
calcium protected the enzyme from WRK modification, heparinase I was
first incubated with 100 mM CaCl2 at room
temperature. Heparinase I digests in the absence of WRK modification
were included as controls.
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RESULTS |
Interaction of Terbium with Heparinase I--
Fig.
1 shows that, in the presence of
millimolar concentrations of calcium, heparinase I activity was
increased 5-fold, consistent with earlier observations (11). To address
whether heparinase I itself binds calcium, we studied the interaction
of Tb3+ with heparinase I in the absence of
heparin. In this way, interactions of heparinase I with terbium could
be studied independently of confounding factors associated with
terbium-heparin interactions. Tb3+ is a lanthanide analog
of calcium often used to probe the nature of protein interactions with
calcium (21, 22). Tb3+ possesses an ionic radius that is
very similar to that of calcium in aqueous solution and has the
advantage that, unlike calcium, the protein-Tb3+ complex is
fluorescent. In addition, because of the increase in charge properties
of terbium compared with calcium (i.e. 3+ as opposed to 2+),
terbium very often has a higher affinity for calcium-binding sites than
does calcium itself.

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Fig. 1.
Heparinase I activity as a function of
calcium concentration. Heparinase I (2 µg) was incubated with 4 mg/ml heparin and various concentrations of calcium in 100 mM MOPS, pH 7.0, at 30 °C. The formation of the
4,5 product was monitored at 232 nm. mAu,
milli-absorbance units.
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Upon titration of heparinase I with terbium, an increase in
fluorescence was observed whether excitation was performed at 488 nm
(direct excitation of the terbium adduct) or 280 nm (excitation of
nearby tyrosine side chains, followed by energy transfer to the terbium
adduct). Since, as has been observed with other protein systems, the
fluorescence signal was enhanced upon indirect excitation at 280 nm,
the most extensive studies were completed in this way (Fig.
2). Fluorescence intensity increased upon
titration of terbium to heparinase I until 10 eq had been added. Beyond
this point, the fluorescence intensity did not increase further.

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Fig. 2.
Terbium binding to heparinase I. Increasing concentrations of terbium were added to heparinase I; the
solution was allowed to come to equilibrium; and the fluorescence at
545 nm was measured with a excitation wavelength of 280 nm. The data
are plotted as relative fluorescence versus terbium/enzyme
equivalents. Maximum fluorescence was achieved upon addition of 10 eq
of terbium.
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To ensure that the terbium-heparinase I interaction was specific, the
ability of calcium to compete with terbium for binding to heparinase I
was investigated. As noted above, terbium very often can have a
1000-fold higher affinity than calcium for calcium-binding sites;
therefore, a large excess of calcium is required to reduce the binding
of terbium to a protein. In the case of heparinase I, after addition of
8 eq of terbium to heparinase I, calcium was added to the
terbium/enzyme solution, and the fluorescence was measured. As shown in
Fig. 3, addition of calcium
concentrations up to 2 mM was able to compete terbium off
of heparinase I. These results indicate that the interaction of terbium
with heparinase I is specific and that this interaction substitutes for
calcium binding to heparinase I.

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Fig. 3.
Calcium competition of terbium-bound
heparinase I. The terbium-heparinase complex was titrated with
increasing amounts of calcium; the sample was allowed to sit for 15 min; and the fluorescence at 545 nm was measured. Fluorescence
intensity decreased with increasing calcium concentration (up to 2 mM).
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Inactivation of Heparinase I with Tb3+ or
Lu3+--
In an effort to confirm and extend the
conclusions of the fluorescence study, the effect of terbium on
heparinase I activity was determined. We found that heparinase I
activity was inhibited in a dose-dependent fashion by
terbium with a measured IC50 of 39 µM (Fig.
4). This type of inhibition has been seen
for other enzyme systems known to interact specifically with calcium
(23). The effect of another lanthanide, lutetium, on heparinase I
activity was also investigated. The ionic radius of Lu3+ is
smaller than that of Tb3+; therefore, we expected that
Lu3+, a less suitable replacement for calcium in heparinase
I, would be a less potent inhibitor of heparinase I activity (24, 25). In fact, Lu3+ was also able to inhibit heparinase I
activity; however, the IC50 was increased to 212 µM. Together with the fluorescence experiments, these
results indicate that heparinase I interacts in a highly specific
manner with terbium and, by extension, with calcium.

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Fig. 4.
Effect of lanthanides on heparinase I
activity. Heparinase I was preincubated with either lutetium ( )
or terbium ( ), and an aliquot of the lanthanide-heparinase I mixture
was added to 4 mg/ml heparin and 5 mM Ca2+ in
10 mM MOPS and 0.1 M KCl, pH 6.5. The activity
of the heparinase I sample was measured by the UV 232 nm assay as
outlined under "Experimental Procedures." mAu,
milli-absorbance units.
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Inactivation of Heparinase I by Chemical Modification--
Having
determined that there is a specific calcium-heparinase I interaction,
we set out to determine which sites in heparinase I could potentially
bind calcium. To investigate the roles of calcium in the enzymatic
activity of heparinase I, we used a combination of chemical
modification coupled with protection experiments (to ensure the
specificity of the reaction) and proteolytic mapping studies (to
identify particular amino acids that are modified). To complete these
studies and to identify the calcium-binding site(s) in heparinase I, we
used two modification reagents (WRK and EDAC) that are specific for
carboxylate groups.
Formation and Degradation of the Ketoketenimine Intermediate from
WRK--
In the case of WRK, the active modification reagent is not
WRK per se, but rather an unstable intermediate, the
corresponding ketoketenimine (19). In an effort to understand the
kinetics of heparinase I modification by WRK, the rates of formation
and degradation of the ketoketenimine were followed by monitoring an
aqueous solution of WRK at 340 nm. At this wavelength, the only species
of WRK that possesses appreciable absorbance is the ketoketenimine (
= 4730 cm
1 mM
1) (19). The
results were fit to a nonlinear equation as outlined under
"Experimental Procedures" (Fig. 5).
The derived rate constants for the formation (0.061 s
1)
and degradation (0.019 s
1) of the ketoketenimine were
used to accurately determine the kinetics of heparinase I inactivation
by WRK.

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Fig. 5.
Formation and decomposition of the
ketoketenimine in MOPS, pH 7.0. The absorbance of a 50 µM aqueous solution of WRK was monitored at 340 nm, and
the amount of the ketoketenimine formed as a function of time was
calculated ( = 4730 cm 1 M 1)
(18, 19). To determine the rate constants of formation (k")
and degradation (k') of the ketoketenimine, Equation 1 (see
"Experimental Procedures") was used.
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WRK and EDAC Inactivate Heparinase I in a
Dose-dependent Way--
Having determined the kinetics of
ketoketenimine formation, the kinetics of WRK inactivation of
heparinase I were studied. WRK was found to inhibit heparinase I in a
dose-dependent fashion (Fig.
6A). Plotting the pseudo
first-order rate constants as a function of the WRK concentration
yielded a second-order rate constant of 7.9 mM
1 min
1 (Fig.
6B).

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Fig. 6.
A, inactivation of heparinase I by WRK.
Heparinase I was incubated with 100 µM ( ), 150 µM ( ), 200 µM ( ), or 400 µM ( ) WRK. The activity at various times was measured
by withdrawing an aliquot and measuring the rate of formation of
unsaturated product. The natural log of the fractional activity was
plotted as a function of an adjusted time term to account for
degradation of the ketoketenimine. B, second-order rate
constant for the inactivation of heparinase I at pH 7.0. The pseudo
first-order rate constants were plotted as a function of the WRK
concentration, and the data were fit to a straight line. The measured
rate constant was 7.9 mM 1
min 1.
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To ensure that the reaction was specific for carboxylate-containing
residues, the effect of another carboxylate-specific reagent (EDAC) on
heparinase I activity was determined. Similar to what was seen for WRK,
EDAC (in the millimolar range) was found to inhibit heparinase I in a
dose-dependent fashion (data not shown).
Protection of Heparinase I from WRK-mediated Inactivation by
Calcium or Heparin--
If WRK modifies the calcium-binding domain(s)
of heparinase I, leading to inactivation because of disrupting
interactions critical for proper enzymatic functioning, then
preincubation with calcium, heparin, or both should offer some
protection from inactivation. To determine whether this is the case,
heparinase I was preincubated for 30 min with either heparin or heparin
and 5 mM Ca2+ (Fig.
7A). Heparin was able to
partially protect the enzyme from inactivation; however, preincubation
with heparin and calcium was able to almost completely protect the
enzyme from inactivation. Preincubation with increasing amounts of
calcium was found to protect heparinase I from WRK-mediated
inactivation, with a K0.5 of 980 µM (Fig. 7B). At large calcium concentrations,
calcium alone (kinact = 2.1 min
1)
protected heparinase I about half as well as heparin plus calcium (kinact = 1.2 min
1).

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Fig. 7.
A, WRK modification of heparinase I is
affected by preincubation with either 4 mg/ml heparin solution or 4 mg/ml heparin and 5 mM Ca2+ solution. Before
addition of WRK, heparinase I was incubated for 20 min with either
heparin alone ( ) or heparin plus calcium ( ). A control reaction
was run with no preincubation ( ). B, protection of
heparinase I from WRK modification by preincubation of the enzyme with
calcium. Heparinase I was preincubated with increasing concentrations
of calcium from 0.1 to 20 mM. After 20 min, 50 µM WRK was added, and the activity of heparinase I was
measured as a function of time.
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Mapping of Residues Modified by WRK--
WRK modification of
specific amino acid residues forms covalent adducts that are stable to
proteolytic mapping. In the presence of a suitable nucleophile, such as
glycine methyl ester, the WRK-carboxylate adduct absorbs in the near-UV
region (280-320 nm) (19). Therefore, the tryptic map of heparinase I
was monitored at 210, 280, and 320 nm, and peaks with absorbance higher
than that of the controls were collected. Fig.
8 (A and B) shows
the HPLC profile of the tryptic digest of heparinase I incubated with
WRK monitored at 210 and 320 nm. In the chromatogram (Fig.
8B), the peptides eluting at 52 (td 52), 54 (td 54), 56.5 (td 56), 59 (td 59), and 95 min (td 95) were sequenced. The complete
sequences of the peptides are shown in the figure legend, and their
positions within the primary amino acid sequence of heparinase I are
shown in Table I.

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Fig. 8.
A, tryptic map of WRK-modified
heparinase I, monitored at 210 nm. Heparinase I was modified as
outlined under "Experimental Procedures." After addition of glycine
methyl ester, the enzyme was digested with trypsin overnight at
37 °C. The fractions were run on reverse-phase HPLC and monitored at
210 and 320 nm. B, same as A, except monitored at
320 nm. Five peaks were observed. The first, eluting at 52 min (td 52),
has the sequence (K)AIIDNK. The second (td 54) and fourth (td 59) peaks
both were different modification products of the sequence
(K)NIAHDKVEKKDK. The third peak (td 56) has the sequence (R)VNVQADSAK.
The last peak (td 95) has the sequence
(K)FGIYRVGNSTVPVTYNLSGYSETAR.
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Table I
Primary amino acid sequence of heparinase I (minus the leader sequence)
and identification of WRK-modified peptides
Boldface sequences are those that conform to the calcium-chelating
EF-hand motif (Table II). WRK-modified peptides are underlined.
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The late eluting peak (td 95) was found to correspond to the C-terminal
region of heparinase I. Two of the four clustered peaks (td 54 and td
59) were found to correspond to a region of the protein that overlapped
with the primary heparin-binding site of heparinase I (11). The other
two peptides (td 52 and td 56) were found to be small peptides in the
N-terminal region of heparinase I, both of which contain aspartate residues.
Identification of CB-1 and CB-2--
Many studies have aimed at
identifying consensus sequences for calcium-coordinating motifs, and
most of these have focused on a particular calcium-coordinating motif,
the EF-hand, present in many calcium-binding proteins (32, 33). To
provide a framework for this study as well as for the accompanying
site-directed mutagenesis study (14), we set out to determine whether
any of the WRK-labeled peptides conformed to an EF-hand motif.
Table II lists the consensus sequence of
the EF-hand calcium-coordinating motif. The canonical EF-hand consists
of two
-helices interposed by a loop region that contains the
calcium-chelating amino acids. These amino acids, identified as
X, Y, Z, -Y, -X, and -Z in Table II (six ligands, typically in an octahedral
geometry), chelate calcium either through an oxygen atom of a side
chain or through a carbonyl atom of the peptide backbone. Examination of the modified tryptic peptides and comparison of their amino acid
sequence with the EF-hand calcium-chelating consensus sequence (Table
II) indicated that two of these modified peptides (viz. the
C-terminal region of heparinase I and the region proximate to the
heparin-binding site of heparinase I) share similarities with a EF-hand
consensus sequence (30) and could potentially bind calcium.
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Table II
Calcium-coordinating motifs in heparinase I
The consensus sequence was derived from Ref. 32. Note that EF-hand
homology was determined by aligning CB-1 and CB-2 sequences with the
consensus EF-hand sequence. Amino acids 1-12 represent amino acids in
the critical loop region of an EF-hand that are responsible for calcium
chelation. Specific chelating amino acids are labeled X,
Y, Z, -Y, -X, and
-Z. Boldface amino acids in the CB-1 and CB-2 sequences
indicate amino acids that conform to the canonical EF-hand motif.
Within the consensus sequence, boldface letters indicate the amino
acids that have been observed among calcium binding proteins in
decreasing order of frequency.
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The first site, hereafter referred to as CB-1, extends from
Glu207 to Ala219 (Table II) and is proximate to
the heparin-binding site that has also been shown to be critical for
enzymatic functioning (11) and that contains His203, a
putative active-site residue (8). Within CB-1, the potential calcium-chelating amino acids include Glu207,
Asp210, Asp212, and Thr216. The
second site, hereafter referred to as CB-2, is at the C terminus of
heparinase I, extending from Thr373 to Arg384.
Like CB-1, CB-2 contains amino acids that could potentially bind
calcium. These include Thr373, Asn375,
Ser377, Ser380, and Glu381. More
importantly, both CB-1 and CB-2 are modified by WRK/glycine methyl ester.
To determine whether these peptides that were modified by WRK in the
above experiment could be protected upon preincubation with calcium,
100 mM Ca2+ was added to heparinase I before
addition of WRK and subsequent digestion. Under these conditions, td 54 and td 59 (corresponding to modification of CB-1) and td 95 (corresponding to modification of CB-2) were all protected from
modification by preincubation with calcium, consistent with CB-1, CB-2,
or both being involved in calcium binding by heparinase I.
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DISCUSSION |
By a combination of biophysical and biochemical techniques, we
have conclusively determined that heparinase I binds calcium. Furthermore, we have shown that the interaction between calcium and
heparinase I is important for proper functioning of the enzyme, and we
have mapped the calcium-binding regions of heparinase I.
Fluorescence titration experiments have often been used to establish a
specific interaction between calcium and a protein, including enzymes.
We found that, in the presence of heparinase I, there is a fluorescence
enhancement of terbium. This enhancement plateaus at a terbium/enzyme
ratio of 10:1. To confirm that this interaction is specific for
calcium, we found that terbium binding can be competed off by addition
of an excess of calcium. Furthermore, terbium has a more pronounced
effect on heparinase I activity than lutetium, a lanthanide with an
ionic radius that does not as closely mimic that of calcium. Once
again, by analogy to other enzyme systems, most notably trypsin (26),
this finding supports the notion of a specific calcium-heparinase
interaction being important for enzymatic activity.
To corroborate the findings of the terbium study, we studied the
interaction of WRK with heparinase I. We found that heparinase I is
inhibited by WRK in a dose-dependent manner, suggesting
that WRK modifies carboxylate residues important for proper enzymatic functioning. WRK is well established in terms of its ability to modify
glutamate and carboxylate amino acids (27-29), which are especially
prevalent in calcium-coordinating motifs. However, recent reports have
highlighted the fact WRK can be nonspecific (30, 31). In fact, WRK can
possibly react with other exposed nucleophiles, including
surface-accessible histidines or cysteines. This was especially of
concern in our case since it is known, from a previous study, that
heparinase I contains an unusually nucleophilic cysteine residue,
cysteine 135 (7).
Several lines of evidence support the supposition that WRK modifies
carboxylate residues in heparinase I. First, preincubation with calcium
was found to protect heparinase I from inactivation by WRK in a
dose-dependent fashion. Second, EDAC, like WRK, was found
to modify heparinase I. Finally, we mapped the residues that were
modified by WRK. These mapping studies revealed that all the peptides
contained carboxylate-containing amino acids. In addition, no peptides
contained cysteine 135, indicating that, under these reaction
conditions, WRK is specific for carboxylate-containing amino acids.
Together, these findings support the notion that WRK modifies
carboxylate-containing amino acids in the calcium-binding motifs of
heparinase I.
The protection data also clearly highlight another point, that in the
enzymatic function of heparinase I, a ternary complex forms between
heparin, calcium, and heparinase I. Preincubation with heparin and
calcium protects heparinase I from modification by WRK almost entirely,
whereas preincubation with either alone does not. Experiments in
progress are aimed at addressing what role this ternary complex plays
in heparinase activity (i.e. whether this complex is
involved in enzyme stability, in the enzymatic cycle of heparinase I,
or in some other necessary process).
Thus, taken together, the chemical modification and fluorescence data
clearly show that calcium binds to heparinase I and that this
interaction is critical for proper functioning of heparinase I. In
addition, the mapping studies implicate two sites on heparinase I that
could potentially bind calcium, either one or both of which are
critical for complete enzymatic activity. Both of the sites contain a
number of amino acids with oxygen-containing side chains, especially
glutamate and aspartate, the preferred chelating motifs for the hard
acid Ca2+ (32, 33).
In summary, the experiments outlined in this study have shown that
heparinase I binds calcium and have identified two sites (CB-1 and
CB-2) that play a role in calcium binding and mediating heparinase I
activity. Thus, this study provides a framework for the mutagenesis
work outlined in the accompanying study (14). There, we set out,
through a rigorous site-directed mutagenesis study, to delineate the
importance of residues in CB-1 and CB-2 for the activity of heparinase I.