(Received for publication, May 3, 1995; and in revised form, May 16, 1995)
From the
Five synthetic substrates containing different amino acid
residues at the P Lysosomal cathepsin B (EC 3.4.22.1), a cysteine proteinase
normally active in protein turnover, has been implicated in several
pathological conditions, including arthritis, muscular dystrophy, and
tumor
metastasis(1, 2, 3, 4, 5, 6, 7) .
It is unique among the cysteine proteinases in that it has both
endopeptidase and exopeptidase activities, can accept basic residues in
a substrate at both the P Kinetic analyses with N-terminal protected
monopeptidyl and dipeptidyl substrates or various irreversible
inhibitors demonstrated that the S In contrast to the
understanding of the S In the present paper we describe the synthesis and
kinetic analysis of substrates of the type
acetyl-X-Arg-Arg-AMC
Assays
were performed in 25 mM sodium phosphate buffer, 250 mM NaCl, 1 mM EDTA, 3% dimethyl sulfoxide, pH 6.0 or 7.7,
and in 25 mM sodium citrate buffer, 250 mM NaCl, 1
mM EDTA, 3% dimethyl sulfoxide, pH 4.0. The pH-activity
profiles were determined for two substrates, acetyl-Val-Arg-Arg-AMC and
acetyl-Arg-Arg-Arg-AMC. These pH-activity profiles were determined over
the pH range 3.2-8.4 by measuring k The citrate buffer consistently afforded lower activity measurements
when compared with the phosphate buffer through the pH range
5.7-6.0, and for this reason there was a break in the continuity
of the data of the pH-activity profile. Similar results were observed
by Hasnain et al.(12) for the substrates
benzyloxycarbonyl-Arg-Arg-X (where X is AMC or p-nitroanilide). The data in citrate buffer were corrected by
a factor determined from measurements in phosphate buffer at four
separate pH intervals (5.7-6.0) where citrate and phosphate
buffers overlapped, as described by Hasnain et
al.(12) . For the tripeptide substrates in this
investigation, a similar effect was evident at pH 7.7, where borate and
phosphate buffers overlapped, with greater activity in borate buffer.
It was determined previously (12) that pH 7.7 was the only
value in the pH range where both phosphate and borate buffers could be
made to overlap under the ionic strength conditions used in the
experiment. Therefore, the following strategy was used to correct for
this buffer effect. The enzyme activity measurements at pH 7.7 for the
substrate acetyl-Val-Arg-Arg-AMC in each buffer system were carefully
repeated in triplicate and were shown to be highly reproducible. By
averaging the enzyme activities in each buffer system at pH 7.7 and by
calculating the ratio of these averaged activities for both buffer
systems, a normalizing factor with small standard deviation was
determined. Graphical analysis of the corrected data for two
independent experiments using the substrate acetyl-Val-Arg-Arg-AMC and
for one experiment using the substrate acetyl-Arg-Arg-Arg-AMC
demonstrated that the profiles gave a best fit to the four-proton
ionization model of Hasnain et al.(12) ,
where dipeptide AMC substrates with an arginyl residue in the P
The
substrates acetyl-Arg-Arg-AMC, acetyl-Gly-Arg-Arg-AMC,
acetyl-Glu-Arg-Arg-AMC, and acetyl-Tyr-Arg-Arg-AMC were prepared in
solution by the employment of N All substrates were purified by
reverse-phase high performance liquid chromatography using varied
linear gradients between 0 and 60% acetonitrile, in 0.1% (v/v)
trifluoroacetic acid. Substrate composition and purity were verified by
amino acid, mass spectral, and
To
refine these enzyme-substrate complexes, low energy conformers were
generated using simulating annealing followed by energy minimization.
Each complex was subjected to 20 simulating annealing experiments. In
each experiment, the complex was subjected to a dynamics simulation
during which the temperature varied from 300 to 30 °C over a period
of 3 ps. The time step was 3 fs. These procedures were carried out
using the QXP program developed at Ciba-Geigy
For the tripeptide substrates
at pH 6.0, specificity increased in the order Glu < Gly < Arg
< Val < Tyr, with a 21-fold difference between the glutamate- and
tyrosine-containing substrates. For the two substrates containing
either arginine or valine at P For the tripeptide
substrates in this investigation, with the exception of
acetyl-Glu-Arg-Arg-AMC, the observed drop in the value of k
Figure 1:
pH-activity profiles of recombinant
cathepsin B. The substrates are acetyl-Arg-Arg-Arg-AMC (
Data were fitted to this model according to the equation of
Hasnain et al.(12) . The pH dependence of
cathepsin B-catalyzed hydrolysis of the substrates
acetyl-Arg-Arg-Arg-AMC and acetyl-Val-Arg-Arg-AMC (Fig. 1) was
very similar to that obtained previously for the substrate
Z-Arg-Arg-AMC (12) . The pK
At pH 6.0, a comparison
of the specificity constants for the substrates acetyl-Arg-Arg-AMC and
acetyl-Gly-Arg-Arg-AMC (Table 1) shows virtually no difference.
It appears, therefore, that the acetyl methyl and the atoms forming the
adjacent amide bond in the tripeptide substrate make no significant
contribution to binding. In addition, the x-ray structure of the
cathepsin B-inhibitor complex reveals that the oxycarbonyl moiety
between the pseudo-P
If the Asp The 2-fold increase in specificity of the P
Figure 2:
Possible binding mode of the substrate
acetyl-Tyr-Arg-Arg-AMC. A possible binding mode of the substrate
acetyl-Tyr-Arg-Arg-AMC to cathepsin B was determined by docking the
substrate into a computer model of the active site obtained from the
crystal structure of cathepsin B. The substrate was energy minimized as
described under ``Experimental Procedures,'' using the Amber
force field as implemented by the MACROMODEL software
package.
The P
Figure 3:
Possible binding mode of the substrate
acetyl-Arg-Arg-Arg-AMC. A possible binding mode of the substrate
acetyl-Arg-Arg-Arg-AMC to cathepsin B was determined as described in Fig. 2.
The modeling study for the substrates containing
either a protonated or deprotonated Glu ( Fig. 4and 5) shows
that the side chain methylene carbons of Glu make van der Waals
contacts with the C
Figure 4:
Possible binding mode of the substrate
acetyl-Glu-Arg-Arg-AMC. A possible binding mode of the substrate
acetyl-Glu-Arg-Arg-AMC to cathepsin B, where the Glu side chain
carboxyl is deprotonated, was determined as described in Fig. 2.
To
summarize, the substrate binding in the S
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS AND DISCUSSION
FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES
position (acetyl-X-Arg-Arg-AMC,
where X is Gly, Glu, Arg, Val, and Tyr and where AMC
represents 7-amido-4-methylcoumarin) were used to investigate the
S
subsite specificity of cathepsin B. At pH 6.0, the
specificity constant, k
/K
, for
tripeptide substrate hydrolysis was observed to increase in the order
Glu < Gly < Arg < Val < Tyr. Molecular modeling studies of
substrates containing a P
Glu, Arg, or Tyr covalently bound
as the tetrahedral intermediate to the enzyme suggest that the
specificity for a P
Tyr is because of a favorable
aromatic-aromatic interaction with Tyr
on the enzyme as
well as a possible H bond between the P
Tyr hydroxyl and
the side chain carboxyl of Asp
.
and P
positions, and
has complex pH
dependences(8, 9, 10, 11) . The
detailed analysis of the pH dependence of cathepsin B-catalyzed
hydrolyses has revealed that at least seven groups that dissociate in
the pH range of 3-9 can affect substrate binding and/or
turnover(12) .
subsite prefers
positively charged or straight chained aliphatic amino acids but
exhibits a very poor affinity for negatively charged or bulky aromatic
and branched aliphatic amino
acids(11, 13, 14, 15, 16) .
The S
subsite was shown to exhibit a preference for
phenylalanine and arginine, although the specificity constant, k
/K
, is 7-fold
higher for the former(12) . Interpretation of pH activity
profile data, obtained with substrates containing an arginine at
P
and either an arginine or phenylalanine at P
,
suggested the presence of carboxyl groups in the S
and
S
subsites with pK
values of
5.4 and 5.1, respectively(12) . The identity of the group with
the pK
of 5.1 was confirmed to be
Glu
by the kinetic characterization of the site-directed
mutants, Glu
Gln
and Glu
Ala
(17) .
and S
subsite
specificities, S
specificity remains largely
uncharacterized. Brmme et al.(18) have shown that the binding of a series of N-terminal
succinylated alanine-containing peptide substrates could be improved by
increasing the length of the peptide chain. When comparing tripeptide
and dipeptide alanine substrates, the former demonstrated a 2-fold
increase in the specificity constant k
/K
. In addition,
the S
subsite was shown to accept proline, unlike subsites
S
or S
(18) . Brmme et al.(19) also examined S
and S
specificity using six tripeptide and tetrapeptide coumaryl
substrates. For two of the substrates with valine and phenylalanine as
the P
residue, they found no significant difference in
binding energy. However, since the N-terminal blocking groups were not
the same for the substrates and the P
residues selected did
not have a sufficiently wide range of physicochemical properties, the
optimal specificity of the S
subsite remained unresolved.
In a different approach, Koga et al.(20) digested soluble denatured proteins with cathepsin B.
Fragments arising from endopeptidase activity were analyzed, and
interpretation of the results suggested a preference for the amino
acids glycine, tryptophan, alanine, lysine, isoleucine, and proline at
the P
position of the substrate. Although only qualitative,
the results, excluding the observation of lysine at P
,
supported the possibility of a hydrophobic pocket at the S
subsite.
(
)(where X is Gly, Glu, Arg, Val, or Tyr). These substrates were designed to
define specificity at the S
subsite. In addition, the
change in free energy of binding (
G values) for
these substrates was determined to compare the relative strengths of
binding of the different P
side chains in the S
subsite. Finally, molecular modeling studies were carried out for
the tripeptide substrates containing either Glu (in its protonated and
deprotonated states), Arg, or Tyr in the P
position to gain
further insights into enzyme-substrate interactions.
Enzyme Expression and Purification
The
cDNA for rat cathepsin B was expressed in Saccharomyces cerevisiae as an -factor fusion construct(21) . Yeast starter
cultures were grown in synthetic medium and then were grown in 4-liter
shake flasks as reported before(22, 23) . The
recombinant enzyme was harvested and purified as described
previously(12) .
Enzyme Assays for the Determination of Kinetic
Constants
Prior to kinetic analysis, the enzyme
concentration was determined by active-site thiol titration using the
cysteine proteinase inhibitor E-64 (N-[N-(L-3-trans-carboxyoxiran-2-carbonyl)-L-leucyl]-agmatine)
(Boehringer Mannheim Canada) according to the methodology of Barrett
and Kirschke(11) . Just before conducting experiments for the
determination of kinetic constants, the enzyme, in 50 mM sodium phosphate buffer, pH 6.0, 1 mM EDTA, was activated
for 1 h by the addition of dithiothreitol to a final concentration of
10 mM and then kept on ice(12) . The active enzyme
concentration was shown to be unchanged for the duration of the
experiments. Michaelis-Menten kinetic parameters for the coumaryl
substrates were determined at 24 °C by continuous fluorescence
spectrophotometry, using an SLM Aminco DW2000 spectrophotometer
equipped with the Total Fluorescence Accessory(12) . All data
were fitted by the nonlinear regression data analysis program
Enzfitter, of Leatherbarrow, supplied by Elsevier-Biosoft./K
at 0.1-pH unit
intervals, using the relationship k
/K
= v/([E][S]) when
[S]K
. Assay samples were
monitored to ensure that pH was stable over the course of the reaction.
Over the pH range 3.2-6.0, the reaction buffer consisted of 25
mM sodium citrate, 250 mM NaCl, 1 mM EDTA,
and 3% dimethyl sulfoxide. In the pH range 5.7-7.7, the reaction
buffer was 25 mM sodium phosphate, 250 mM NaCl, 1
mM EDTA, and 3% dimethyl sulfoxide; and in the pH range of
7.7-8.4, the reaction buffer was 25 mM sodium borate,
250 mM NaCl, 1 mM EDTA, and 3% dimethyl sulfoxide.
position were employed. The agreement with previous work
supported the validity of this correction for the borate buffer.
Substrate Synthesis
The peptides
acetyl-Arg-Arg-Arg and acetyl-Val-Arg-Arg were synthesized on a Sasrin
resin solid support by the employment of standard Fmoc methodology. The
side chain functionality of N-Fmoc-arginine
was blocked with the 2,2,5,7,8-pentamethylchroman-6-sulfonyl group.
Amino acids were coupled using HBTU in N-methyl pyrrolidone.
After being cleaved from the resin with 1% trifluoroacetic acid in
dichloromethane, the peptides were coupled to AMC with EDC in
dimethylformamide and dichloromethane, once again in the presence of
1-hydroxybenzotriazole. Deblocking the completed substrate was effected
by treatment with 95% trifluoroacetic acid and 5% water.
-t-Boc
amino acids. In contrast to the solid support method, the initial
syntheses of these substrates began with the making of Arg-AMC, and the
coupling reaction was carried out in pyridine with
dicyclohexylcarbodiimide. The side chain functionalities of Arg, Tyr,
and Glu were protected as p-toluenesulfonyl,
2-bromobenzyloxycarbonyl, and benzyl derivatives, respectively. Peptide
coupling reactions with dicyclohexylcarbodiimide were generally
achieved in a double solvent system. Typically, dichloromethane was
used to dissolve the t-Boc amino acid and coupling agent. To
this was combined the growing AMC peptide, previously dissolved in N,N-dimethylformamide. Racemization was minimized by
the employment of 1-hydroxybenzotriazole. Sequential removal of the t-Boc group required treatment with 50% trifluoroacetic acid
in dichloromethane at room temperature. Substrates were subsequently
acetylated with acetic anhydride in pyridine. Deprotection of the side
groups was effected by treatment with a 9:1 (v/v) mixture of
hydrofluoric acid/anisole.
H NMR spectral analyses.
Evidence Supporting Exclusive Hydrolysis of
Substrates at the AMC Peptide Bond
Exclusive hydrolysis of
substrates at the AMC peptide bond was established by using high
voltage paper electrophoresis to separate the products after incubation
of substrates with cathepsin B. Since AMC is ninhydrin-negative, the
basic premise of this strategy was that cleavage at any site, other
than the AMC peptide bond, would generate a free -amino group,
which would yield a ninhydrin-positive peptide. Substrates were
incubated with cathepsin B in pH 6.5 buffer of the composition acetic
acid/pyridine/water (3:100:1,800 v/v/v). Reaction completion was
verified spectrophotometrically, whereupon the digests were applied to
3MM Whatman paper and subjected to electrophoresis in pH 6.5 buffer
(3:100:900 v/v/v) at a voltage gradient of 60 volts/cm for 30 min. All
substrate digests yielded products that tested ninhydrin-negative,
demonstrating that cleavage occurs only at the AMC peptide bond. After
spraying the chromatogram with the modified Sakaguchi
reagent(24) , arginine residues were identified as a single red
band, indicating that there was only one product containing arginine.
The final position of this fragment and its lack of fluorescence under
ultraviolet light were consistent with the expected behavior of each
substrate after cleavage at the AMC peptide bond. The absence of other
Sakaguchi-positive material also ruled out other possible cleavage
reactions. Standards of unwanted cleavage products were employed at
predetermined concentrations and demonstrated that undesirable digest
fragments would have been detected at the 1 mol % level.
Molecular Modeling of Enzyme-Substrate
Interactions
Modeling studies were carried out for the
tripeptide substrates containing a P Tyr, Arg, or Glu, the
last in the protonated and deprotonated forms. Each substrate was
constructed using, as a starting point, the x-ray structure of the
inhibitor, Pro-Phe-Arg-CMK bound to cathepsin B.
(
)The MACROMODEL molecular modeling package (25) was used for this operation. The substrate was covalently
attached to the enzyme simulating a tetrahedral intermediate by forming
a covalent bond between the active-site thiolate of Cys
and the P
carbonyl carbon of the substrate.
(
)with parameters from the AMBER force
field(26) . Comparison of the positions of the enzyme atoms
from the x-ray structures of cathepsin B complexed with several
different inhibitors showed that the positions of most of the enzyme
atoms did not change significantly with binding of different
inhibitors
(
)(27) . Therefore, most of
the enzyme atoms were held stationary during energy minimization.
Residues that were allowed to move were Gln
,
Cys
, Asp
, Glu
, and
Glu
.
Michaelis-Menten Constants
Table 1lists the substrates and the kinetic constants
for their hydrolysis by cathepsin B. At pH 6.0, k/K
for the
substrate acetyl-Arg-Arg-AMC was 4-fold less than
benzyloxycarbonyl-Arg-Arg-AMC. The difference appears largely due to an
increase in K
, which can be attributed to
the substitution of the benzyloxycarbonyl group by the acetyl group.
This is consistent with the fact that the x-ray structure of the
inhibitor, benzyloxycarbonyl-Arg-Ser-(O-Bzl)-CMK, in a
covalent complex with cathepsin B, reveals a favorable interaction
between the N-terminal benzyl group and the phenyl ring of
Tyr
(27) . This x-ray structure also suggests that
if the binding of the acetyl were analogous to the position of the
oxycarbonyl moiety in the inhibitor's benzyloxycarbonyl group,
the N-terminal acetyl group, in acetyl-Arg-Arg-AMC, would not make any
significant contacts with the enzyme.
, kinetic analysis at pH 7.7
showed that their second-order rate constants increased only slightly
when compared with their values at pH 6.0. However, at pH 4.0, the
second-order rate constants were significantly lower than the
respective values at pH 6.0. The k
/K
values of the
tripeptide substrates at pH 4.0 were generally 15-20-fold lower
than at pH 6.0, the only exception being the result for
acetyl-Glu-Arg-Arg-AMC. This major dependence of k
/K
on pH has been
observed previously for dipeptide substrates of cathepsin B which
contained a P
arginine(12, 17) . The poor
specificity observed for the substrate acetyl-Glu-Arg-Arg-AMC at pH 6.0
is best illustrated by a K
value that is
too large (estimated at greater than 10 mM) to measure. At pH
4.0, however, under which condition the side chain carboxyl group of
the P
glutamyl could be more than 50% protonated (assuming
a pK
of 4.5)(28) , there was a
marked improvement in binding affinity as suggested by the drop in K
compared with the value at pH 6.0. As a
result k
/K
values
for this substrate were very similar at pH 4.0 and 6.0. For all other
tripeptide substrates in this investigation (Table 1), as well as
for the substrate Z-Arg-Arg-AMC(12) , the approximately 20-fold
decrease in the value of k
/K
at pH 4.0, when compared with pH 6.0, is, to a large extent,
due to the protonation state of the Glu
side chain in the
S
subsite of cathepsin B(17) . It was concluded
from the investigation of site-directed mutants that the side chain
carboxylate of the S
Glu
forms a salt bridge
with the guanidinium cation of a P
arginine in dipeptide
substrates, such as Z-Arg-Arg-p-nitroanilide. The strength of
this interaction, which appears to stabilize the transition state
complex, was shown to be weakened by 1.2 kcal/mol upon protonation of
the Glu
side chain carboxylate(17) . This P
Arg-S
Glu
interaction has recently been
confirmed by the determination of the x-ray structure of cathepsin B
complexed with the inhibitor
Z-Arg-Ser-(O-Bzl)-CMK(27) .
and k
/K
at pH 4.0,
compared with pH 6.0 (Table 1), may similarly be explained by a
weakening of the P
-S
interaction upon the
protonation of the Glu
side chain carboxylate. Any drop
in k
/K
which may
have been expected for acetyl-Glu-Arg-Arg-AMC at pH 4.0 relative to pH
6.0, due to the P
-S
interaction, appears to
have been compensated for by an increase in k
/K
because of an
improved P
-S
interaction. Indeed, if the data
are normalized to account for the 20-fold drop normally observed at pH
4.0 relative to pH 6.0 for substrates with a P
Arg and a
neutral P
, there is an approximate 20-fold increase in
affinity for the protonated side chain carboxyl of the P
Glu when compared with the deprotonated form.
Analysis of pH-Activity Profiles for the Substrates
Acetyl-Arg-Arg-Arg-AMC and Acetyl-Val-Arg-Arg-AMC
The data for the pH-activity profiles (Fig. 1) were
best fitted to a model involving two dissociation events in the
ascending limb and two dissociation events in the descending limb as
described before for the dipeptide substrate
Z-Arg-Arg-AMC(12) . EH and E are
inactive forms of the enzyme.
) and
acetyl-Val-Arg-Arg-AMC (
). The lines through the data
points represent the best fit using equations previously described. A
comparison with the substrate Z-Arg-Arg-AMC (-) (12) is
shown.
values for the former tripeptide substrates were in close
agreement with the values for the dipeptide substrate (Table 2).
The greater uncertainty in the values for
pK
2 and pK
3
were due, in part, to the difficulty of obtaining accurate initial
rates above pH 7.9, caused by the pH instability of cathepsin B in that
pH range. These data suggest that the interaction of the P
Arg in the S
subsite is not to any significant degree
a result of a charge-charge interaction, although a weak interaction
cannot be ruled out.
Nature of the S
Table 3reports the change in apparent binding energies
among the dipeptide and tripeptide substrates at pH 6.0. Results are
summarized as follows.-P
Interaction
(i) Apparent Binding Energies for the Dipeptide Substrates
Acetyl-Arg-Arg-AMC and Z-Arg-Arg-AMC
The change in apparent
binding energy for the substrate acetyl-Arg-Arg-AMC relative to
Z-Arg-Arg-AMC was calculated to be 0.82 kcal/mol, suggesting that the
benzyloxycarbonyl group interacts with the enzyme as a pseudo-P residue. As mentioned previously, this finding is supported by
the x-ray structure of a cathepsin B-inhibitor complex (27) .
In this complex, the benzyl ring of the N-terminal benzyloxycarbonyl
group of the inhibitor, Z-Arg-Ser-(O-Bzl)-CMK, makes a direct
contact with the enzyme, forming a vertical aromatic-aromatic
interaction with Tyr
, with the shortest distance of about
3.71 Å.
(ii) Binding Energies of Various P
The glycyl-containing substrate served as
the reference for the calculation of apparent binding energies of the
PSide Chains
side chains of the other tripeptide substrates (Table 3). At pH 6.0, the only P
side chain that
demonstrated a weaker binding energy relative to glycyl was glutamyl,
with a
G of 0.33 kcal/mol. The other three
tripeptide substrates, with either Arg, Val, or Tyr at P
,
exhibited an improved binding in that order. Although the substrate
containing a P
arginyl has an increased binding energy
compared with glycyl, by about 0.81 kcal/mol, this enhancement cannot
be to any large degree due to a charge-charge interaction at the
S
subsite. First, a 4-fold effect arising from an ionic
interaction between the P
guanidinium group and a
negatively charged group in the S
subsite should have been
manifested in the pH-activity profile. As shown in Fig. 1and Table 2, the pH-activity profile data for the tripeptide
acetyl-Arg-Arg-Arg-AMC could be fitted by the same equation used to fit
the data for the dipeptide Z-Arg-Arg-AMC, and the substrate with a
neutral P
, acetyl-Val-Arg-Arg-AMC. Furthermore, the
dissociation constants derived from these data were virtually
identical. In fact, the ratios of k
/K
of
acetyl-Arg-Arg-Arg-AMC and acetyl-Val-Arg-Arg-AMC are similar at pH 6.0
and 4.0. This is also true for the ratios of k
/K
of
acetyl-Arg-Arg-Arg-AMC and the tripeptide substrates with either glycyl
or tyrosyl side chains at P
, which suggests that the
P
arginyl side chain binding is not to any significant
extent stabilized by an ionic interaction. The x-ray structure of the
complex of cathepsin B and the inhibitor Z-Arg-Ser-(O-Bzl)-CMK
suggests a relatively hydrophobic S
pocket defined by
Tyr
, C
of Gly
, C
of
Asn
, and C
of Asp
(27) .
Therefore one may suggest that the improved binding of arginine over
glycine at P
may be largely due to hydrophobic interactions
of the methylene groups in the arginine side chain with the relatively
hydrophobic S
subsite. An interaction between the
Asp
side chain carboxylate and the P
guanidinium cannot be entirely ruled out. However, if it occurs,
this interaction does not contribute very significantly to substrate
binding (see below for further discussion).
N-terminal benzyl group and the
P
residue does not make any contact with the
enzyme(27) . As such, the values for the change in free energy
of binding (Table 3) for different tripeptide substrates also
serve to define the incremental increase in binding energy of the
P
residue when compared with the dipeptide substrate,
acetyl-Arg-Arg-AMC.
(iii) Evidence Supporting a Predominantly Hydrophobic
Pocket at S
From the specificity constants reported
in Table 1and changes in binding energy in Table 3, the
nature of the S subsite of cathepsin B can be deduced. The
S
subsite shows a general preference for both aromatic and
aliphatic groups. At pH 6.0, the substrate with the P
glutamyl is least favored, whereas at pH 4.0 it is preferred over
the arginyl and valyl side chains. Therefore, it appears that a
negative charge at P
seems to disrupt the binding at the
S
subsite. This may be due to a charge-charge repulsion
involving Asp
.
carboxylate is
responsible for destabilizing the binding of a P
negative
charge, it is surprising that the 4-fold increase in specificity of the
arginyl side chain compared with glycyl does not appear to result from
a charge-charge attraction with Asp
. A possible
explanation for the 4-fold increase in specificity of the Arg side
chain compared with Gly is the possible van der Waals interaction of
one or more of the arginyl side chain methylene carbons with
Tyr
.
tyrosyl side chain, relative to the P
valyl, and the
4-fold increase in specificity of the P
tyrosyl, relative
to the benzyl ring of the substrate Z-Arg-Arg-AMC, may result from an
improved interaction of the phenyl ring of the substrate tyrosyl and
Tyr
in the S
subsite of the enzyme. In the
x-ray structure of the cathepsin B-inhibitor complex, the benzyl ring
of the benzyloxycarbonyl moiety is about 3.7 Å from the
Tyr
phenyl ring. The kinetic data suggest that both the
valyl and tyrosyl side chains in the respective substrates make
energetically more favorable contacts with Tyr
than the
benzyl ring of the benzyloxycarbonyl moiety.
Modeling Tripeptide Substrates Containing Tyr,
Glu, or Arg at P
The modeling study with the substrate containing a P Tyr (Fig. 2) revealed that the phenyl ring of the
substrate is partly stacked on the phenyl of Tyr
of the
enzyme, with the shortest distance between C
of the substrate Tyr
and C
of Tyr
of 3.4 Å. In the model, the
substrate tyrosyl hydroxyl forms a hydrogen bond with Asp
.
Another potential contact in the S
pocket is between the
substrate Tyr C
and C
of Gly
(3.6 Å). The
improved contact of the P
tyrosyl in the S
subsite, as suggested by the model, relative to the position of
the pseudo-P
benzyl group of the dipeptide inhibitor
observed in the x-ray structure(27) , is consistent with the
kinetic data showing a 4-fold increase in specificity for the P
Tyr compared with the N-terminal benzyloxycarbonyl group. A study
by Serrano et al.(29) revealed that
aromatic-aromatic interactions in protein structures can contribute
between 0.6 and 1.3 kcal/mol to protein stability. This effect is due
to a quadrupole-quadrupole interaction between the aromatic rings, for
which there is an associated potential energy that varies as
1/r
(where r is the quadrupole separation
distance)(30) . Therefore, any binding energy that is
contributed by aromatic side chains of substrates or inhibitors
interacting with aromatics on enzymes would be very sensitive to the
distance between the groups.
Arg modeling study (Fig. 3) reveals that the methylene groups of the arginyl side
chain are somewhat further from Gly
(4.3 Å) and
Tyr
(4.4 Å) than the other modeled substrates,
suggesting that a van der Waals interaction is unlikely. The
guanidinium group of the P
Arg appears to form hydrogen
bonds with the hydroxyl of Tyr
(3.0 Å) and the
carboxyl oxygen of Asp
(2.7 Å) and, possibly, a weak
hydrogen bond with the backbone carbonyl oxygen of Asn
(3.4 Å). As discussed before, the kinetic data suggest that
if there is an interaction between the guanidinium moiety of the
P
Arg and the carboxylate of Asp
, there is no
significant net gain in binding energy. Considering the fact that there
would be a significant energy cost involved in desolvating the
guanidinium cation and that this interaction would be completely
solvent-exposed, it may be reasonable to expect that there may not be
any significant gain in binding energy for this charge-charge
interaction. Therefore, the 4-fold increase in specificity observed for
a P
Arg, relative to Gly, may be due instead to the
charge-neutral hydrogen bonds involving the substrate guanidinium and
the hydroxyl of Tyr
and possibly the carbonyl of
Asn
.
of Gly
(3.2 Å) and the
phenyl ring of Tyr
(3.6 Å). The kinetic data show
that k
/K
drops
20-fold when the P
Glu deprotonates. One possible
explanation of this significant destabilization of binding may be a
charge-charge repulsion between Asp
and the P
side chain carboxylate, which are about 4.4 Å apart. Since
the potential energy of charge-charge interactions varies as 1/r (where r is the charge separation), they can be
manifested over relatively large distances. In fact, the modeling study
with the deprotonated Glu shows the Asp
side chain moving
away from the P
carboxylate relative to the Asp
side chain position in the modeling studies with the other
substrates. Another possible explanation, however, may be that there is
a repulsion between the
-electron cloud of Tyr
and
the negative charge on the substrate side chain (30) .
subsite of
cathepsin B is largely due to contacts with the phenyl ring of
Tyr
and to a lesser extent may involve the C
of
Gly
. Additionally, hydrogen bonds involving the Tyr
hydroxyl and the main chain carbonyl oxygen of Asn
may also make a contribution to substrate stabilization. The best
substrate among the tripeptides tested has tyrosine at P
,
which in comparison with the reference substrate containing glycine,
has an increase in binding energy of 1.5 kcal/mol. Although these
findings are significant for inhibitor design strategies, it may be
useful to examine further the S
specificity of cathepsin B
with substrates containing residues such as tryptophan, phenylalanine,
proline, methionine, leucine, and isoleucine at the P
position. The study of these additional residues would provide a
more complete understanding of the S
specificity pocket.
Extension of such a study to S
-P
interactions
will contribute additional information for the design of more specific
inhibitors.
We are indebted to A. Szabo for the use of his
laboratory facilities and to A. Wigg for assistance.
©1995 by The American Society for Biochemistry and Molecular Biology, Inc.