(Received for publication, January 5, 1995; and in revised form, May 10, 1995)
From the
The role of the asparagine residue in the Cys-His-Asn
``catalytic triad'' of cysteine proteases has been
investigated by replacing Asn
Cysteine proteases are a class of enzymes requiring the thiol
group of a cysteine residue for their catalytic activity(1) .
The additional involvement of an histidine residue in the catalytic
process was inferred on kinetic grounds(2) , and evidence for
the location of an histidine in proximity to the catalytic thiol group
was provided initially by the use of a bifunctional irreversible
inhibitor of papain (3). The Cys
With the availability of the three-dimensional
structure, other residues were found in the vicinity of the active site
that could possibly play important roles in the mechanism of the
enzyme. In particular, an asparagine residue that is conserved in all
cysteine protease sequences of the papain family, Asn
Figure 1:
Schematic
representation of the active site of papain showing the catalytic triad
residues Cys
There has been no quantitative experimental study addressing the
role of the asparagine residue in the catalytic triad of cysteine
proteases. In a preliminary study using random mutagenesis and
screening of mutants, we have shown that replacement of Asn
On-line formulae not verified for accuracy
On-line formulae not verified for accuracy REACTION 1
On-line formulae not verified for accuracy
In this equation, (k
Figure 2:
Segregation of propapain mutants between
pellet and supernatant and sensitivity to proteolytic degradation by
subtilisin. Panel A, autoradiogram of Western blot for the
pellet fraction (P) and the supernatant fraction (S)
before (-) and after (+) treatment with subtilisin. The
source of the sample is indicated above each series of four samples.
The location of mature papain and propapain is indicated in the rightmargin of the autoradiogram. Panel B,
histogram representing the relative levels of insoluble (P-) to soluble (S-)
proenzymes.
Figure 3:
Kinetics of thermal inactivation of mature
papain. Partially purified preparations of WT (
The influence of pH on (k
Figure 4:
pH dependence of (k
The model introduced to establish the relationship between the
stability of the thiolate-imidazolium ion pair and the measured kinetic
parameters has not been applied so far to the characterization of
mutations involving directly one of the active site residues
(Cys
On-line formulae not verified for accuracy
Figure 5:
Model describing the ionization pathways
for the active site residues Cys
Figure 6:
Simulated curves illustrating the
relationships between kinetic parameters and ion pair stability. Panel A, relationship between the variation in width of a pH
activity profile (
On-line formulae not verified for accuracy
where f represents the effect of a mutation on the
intrinsic activity of the enzyme (i.e. the ratio of the
intrinsic k
To account for the possibility that (
For the Asn
The role of the asparagine residue in the Cys-His-Asn
``catalytic triad'' of cysteine proteases has been
investigated by replacing Asn
A significant
fraction of papain exists with the Cys
As discussed above, it is difficult to dissect out
the relative effect of the mutation on ion pair stability and intrinsic
activity for the Asn
The geometry of the active site Cys-His-Asn residues in
cysteine proteases is very similar to that of the corresponding
Ser-His-Asp residues forming the catalytic triad of serine
proteases(30) . Replacement of the Asn
The magnitude of the
apparent contribution to enzymatic activity of the Asn residue in
cysteine proteases and the corresponding Asp residue in serine
proteases may reflect basic differences in the catalytic mechanism of
the two classes of enzymes(35) . In the case of serine
proteases, the formation of the transition state and tetrahedral
intermediate is accompanied by charge separation, and it has been
suggested that the negative charge on the aspartate can help this
process through electrostatic stabilization, therefore contributing to
catalysis(36) . In cysteine proteases, the Asn residue in the
catalytic triad might be of importance for stabilizing the ion pair
form of the catalytic residues (i.e. the ground state of the
enzyme) by contributing to maintain the active site residues in a
favorable geometry. In contrast to serine proteases, charge separation
is already present in the ground state and generation of the transition
state and tetrahedral intermediate causes only a rearrangement of the
charges. In addition, the Asn residue could play a role in catalysis
through the orientational effect of the H bond to His
Highly conserved residues at the
active site of enzymes are often regarded as being essential for
activity. For the cysteine proteases, it is difficult to account for
the strict conservation of Asn
in papain by alanine and
glutamine using site-directed mutagenesis. The mutants were expressed
in yeast and kinetic parameters determined against the substrate
carbobenzoxy-L-phenylalanyl-(7-amino-4-methylcoumarinyl)-L-arginine.
At the optimal pH of 6.5, the specificity constant (k
/K
)
was reduced by factors of 3.4 and 150 for the Asn
Gln and Asn
Ala mutants,
respectively. Most of this effect was the result of a decrease in k
, as neither mutation significantly affected K
. Substrate hydrolysis by these mutants is
still much faster than the non-catalytic rate, and therefore
Asn
cannot be considered as an essential catalytic
residue in the cysteine protease papain. Detailed analyses of the pH
activity profiles for both mutants allow the evaluation of the role of
the Asn
side chain on the stability of the active site
ion pair and on the intrinsic activity of the enzyme. Alteration of the
side chain at position 175 was also found to increase aggregation and
proteolytic susceptibility of the proenzyme and to affect the thermal
stability of the mature enzyme, reflecting a contribution of the
asparagine residue to the structural integrity of papain. The strict
conservation of Asn
in cysteine proteases might therefore
result from a combination of functional and structural constraints.
-His
arrangement in the catalytic center of papain was established
when the three-dimensional structure of the enzyme was
solved(4, 5, 6) . The papain molecule is folded
to form two interacting domains delimiting a cleft at the surface of
the enzyme. Cys
and His
are located at the
interface of this cleft on opposite domains of the molecule; Cys
is part of the L1
-helix at the surface of the left domain,
while His
is in a
-sheet at the surface of the right
domain of the enzyme.
,
was found to be adjacent to the catalytic His
residue.
The amide oxygen of the Asn
side chain is hydrogen-bonded
to N
H of His
creating a
Cys-His-Asn triad, which can be considered as being analogous with the
Ser-His-Asp triad of serine proteases (Fig. 1). The side chain of
Asn
is buried in a hydrophobic region of the enzyme
composed mainly of residues Phe
, Val
,
Trp
, and Trp
. Residues 141, 177, and 181
are located near the Asn
-His
hydrogen bond
and can shield it from the external solvent. An important feature of
the Asn
-His
interaction is that the
hydrogen bond is approximately colinear with the His
C
-C
bond, allowing rotation of
the imidazole ring about the C
-C
bond
without disruption of the Asn
-His
hydrogen
bond. Comparison of results from crystallographic studies with various
forms of papain either free or alkylated at the Cys
sulfur
atom by chloromethyl ketone inhibitors have demonstrated that the
His
side chain can change its orientation by about
30° (7). Therefore, it has been suggested that the role of
Asn
is to orient the His
side chain in the
optimum positions for various steps of the catalytic mechanism. In the
resting state of the enzyme, the His side chain would be coplanar to
the Cys
residue while during acylation, the protonated
imidazole ring would rotate to act as a proton donor to the nitrogen
atom of the leaving group of the substrate(8) .
, His
, and Asn
.
The representation is derived from the crystal structure of Drenth et al. (7). In the crystal structure, the active site cysteine
is oxidized, and therefore the precise relative orientations of the
Cys
and His
side chains in the non-oxidized
enzyme might differ from the illustrated
orientations.
An important
feature of papain and other cysteine proteases in general is the high
nucleophilicity of the sulfur atom of the active site cysteine residue.
This is due to the fact that at the pH values where the enzyme is
active, the sulfur atom is present as a thiolate anion. It is now
generally accepted that the side chains of Cys and
His
possess unusual pK
values and that the active form of the enzyme consists of a
thiolate-imidazolium ion pair at neutral
pH(9, 10, 11, 12) . However, the nature
and significance of the factors that are responsible for the formation
and maintenance of the ion pair within a wide range of pH for the most
part remain unknown, and this aspect has been the object of many
theoretical studies over the years (see, e.g., Refs.
13-19). Since the side chains of Asn
and
His
interact directly via hydrogen bonding, one of the
obvious roles of Asn
could be to stabilize the
thiolate-imidazolium form of papain. It has been suggested that the
proximity of the active site cysteine and histidine residues could be
one of the most important factors contributing to the formation of an
ion pair and that the proton affinities of Cys
and
His
at the active site of papain are strongly sensitive
to the geometry of these residues(17, 19) .
Consequently, Asn
could stabilize the ion pair by keeping
the imidazole ring of His
in a favorable orientation.
by several amino acids results in a significant loss of
activity(20) . However, due to the relatively low sensitivity of
the assay, this system can unambiguously detect only mutants with
activity similar to wild-type papain. In addition, enzyme inactivation
could occur for mutant enzymes that have a decreased stability under
the relatively drastic conditions used to activate the enzyme
precursors (low pH and high temperature). The screening system we used
cannot readily distinguish between a decrease in catalytic activity and
a decrease in protein stability. In this study, the role of Asn
at the active site of cysteine proteases was investigated by a
detailed kinetic and functional characterization of papain mutants.
Mutation of Asn
to a glutamine was chosen due to our
previous observation that this mutation generates an enzyme that
retains some activity(20) , indicating that the conservative
substitution of Asn
by Gln is tolerated in the active
site of papain. Complete removal of the hydrogen bonding capability of
the side chain of residue 175 was accomplished by an Asn
Ala change.
Expression and Purification of Papain
Mutants
Expression of wild-type papain and of the Asn
Gln and Asn
Ala mutant proenzymes in Saccharomyces cerevisiae has been reported
recently(20) . Yeast cells from 1 liter of culture (8
10
cells/ml) were collected by centrifugation and
resuspended in 20 ml of 10 mM Tris-HCl, pH 7.5, 1 mM EDTA to yield a final volume of about 35 ml. The cells were lysed
using a French press (20,000 p.s.i.) and the cellular debris removed by
a 10-min, 15,000
g centrifugation. Propapain present
in the supernatant was converted to mature papain by limited
proteolysis with subtilisin BPN` (Sigma). The soluble extract was
incubated for 2-3 h at 37 °C in the presence of 0.1 mg/ml
subtilisin. The extract was then changed to pH 5.0 with sodium acetate
buffer (30 mM, pH 4.0) and incubated at 55 °C for 15 min.
After a 10-min centrifugation at 15,000
g,
precipitated proteins were discarded and the supernatant was made 80%
ammonium sulfate and kept at 4 °C overnight. This suspension was
centrifuged at 22,000
g for 20 min and the protein
pellet resuspended in 4 ml of 100 mM sodium acetate, 1
mM EDTA, pH 5.3. This preparation was used to determine the
protein half-life (see below). The enzymes used for the kinetic
characterization were further purified by covalent chromatography using
a thiopropyl-Sepharose column(21) .
Kinetics of Irreversible Thermal Inactivation
The
kinetics of irreversible thermal inactivation of papain variants was
determined as described previously(22) . Partially purified
papain preparations (see above) were adjusted to pH 6.0 with 100
mM phosphate buffer and HgCl added to 5
mM. They were incubated at 82 °C for 0-60 min, and
the residual papain activity was measured. The TI
value
(the time at which the enzyme has lost half of its activity) was
determined from the slope of the linearized form of the
data(22) .
Aggregate/Soluble Precursor Partitioning and
Susceptibility to Protease Degradation
Total yeast extracts (3
ml) were prepared from 75 ml of culture grown under the conditions
defined above. Processing of propapain was prevented with 0.1
mM of E-64
(1-[[(L-trans-epoxysuccinyl)-L-leucyl]amino]-4-guanidino)butane)
(23). The extract was deglycosylated by incubation for 2 h at 37 °C
in the presence of 35 mM sodium acetate buffer, pH 5.5, 200
mM -mercaptoethanol, and 50 milliunits/ml endoglycosidase
H (Boehringer Mannheim). An aliquot of the mixture was centrifuged at
15,000
g for 5 min. The supernatant was recovered and
the pellet was resuspended in 200 µl of phosphate buffer, pH 6.5.
An aliquot of the pellet and supernatant deglycosylated fractions was
analyzed by Western blot prior to or following incubation with 0.1
mg/ml subtilisin for 2 h at 37 °C. Quantitative Western blot
analyses were performed using two rounds of antigen detection after
separation of the proteins in SDS-polyacrylamide gel electrophoresis.
Mature and proenzyme forms of papain were detected with an anti-papain
rabbit polyclonal antibody (24). Papain-antibody complexes were labeled
with I
-labeled protein A (Amersham Corp.) and visualized
by autoradiography. The antigen was then stained in a second reaction
using alkaline phosphatase-conjugated goat anti-rabbit IgGs (Bio-Rad).
This procedure facilitates accurate cutting of the immunoreactive bands
for radioactivity measurements using an LKB 1282 Compugamma counter.
Kinetic Measurements
The kinetic parameters were
obtained as described previously(21) . The concentration of
active purified enzyme was determined by titration with
E-64(25) .
Carbobenzoxy-L-phenylalanyl-(7-amino-4-methylcoumarinyl)-L-arginine
(Cbz-Phe-Arg-MCA)(
)wasused as a substrate. The
reaction conditions consisted of 50 mM phosphate buffer, 0.2 M NaCl, 5 mM EDTA, 10% CH
CN, pH 6.5. For
the determination of pH activity profiles, 50 mM citrate or 50
mM borate were also used as buffers and the substrate
concentration was kept well below the K
value. Kinetic parameters at optimum pH (6.5) were
determined by linear regression of the initial rate (v) data
to plots of s/vversuss (Hanes
plots). The pH activity profiles were analyzed according to the model
of by nonlinear regression of the data to the
corresponding equation (Equation 1).
/K
)
represents the experimentally determined value of the specificity
constant and (k
/K
)
is the limiting value determined from nonlinear regression.
Computer Modeling
Computer modeling was used with
the Asn
Gln mutant to predict the orientation of
the Gln
side chain. The model representing free papain
was obtained using the coordinates from the crystal structure of Drenth et al.(7) . In the model, the oxygen atoms on the
oxidized Cys
residue were removed, and AMBER partial
charges were assigned considering that the active site residues are
present in the thiolate-imidazolium ion pair state. In an initial step,
the Systematic Search module of Sybyl 6.0 (Tripos Associates, Inc.) was
used to carry out a search for sterically allowed conformations of the
Asn
Gln mutant. The Asn
residue was
replaced by Gln and the side chain angles
,
, and
of Gln
were
varied by 2-degree increments. Two ``groups'' of structures
(structure 1 and structure 2) were found, both containing an hydrogen
bond between the oxygen atom of the Gln
side chain amide
and N
H of His
. The structures differ by
the positioning of the acetamide NH
group, which can be
either in proximity of Trp
and Trp
(structure 1) or oriented toward the interface between the two
domains of papain where it can form a hydrogen bond with the side chain
of Ser
(structure 2). Each one of these two conformations
was energy-minimized in an attempt to predict if one orientation would
be favored over the other. Conformational energies were calculated
using the AMBER force field and partial charges. A distance-dependent
dielectric constant,
= r, was used with a
residue-based cutoff distance of 8.0 Å. The minimization was
carried out to a root mean square gradient of 0.05. Energy calculations
were performed on a region delimited by a 12-Å sphere around the
C
atom of Gln
, while atoms within a
9-Å sphere around the same C
atom were allowed to
move during the minimization. The difference in energy between the two
minimized structures was 2.0 kcal/mol in favor of structure 1. However,
this value is close to the precision of the calculations and the second
conformational state (structure 2) cannot clearly be ruled out. For
structure 1, the torsional angles of the Gln
side chain
are
= -115.77,
= 160.84,
= -119.87,
= -85.88,
and
= 77.61, and the Gln
O
-His
N
H distance
is 2.77 Å. With this conformation, the acetamide H atoms of
Gln
are positioned to interact with the
clouds of
the two tryptophan residues (Trp
and Trp
).
In the alternate structure (structure 2),
=
-116.53,
= -179.33,
= -120.38,
= -67.86,
and
= -92.45, and the Gln
O
-His
N
H distance
is 2.76 Å.
Pellet/Supernatant Partitioning and Protease
Susceptibility of Propapain
We have investigated the
consequences of replacing Asn upon the ability of the
protein to be detected as a molecule with native properties. During the
course of purifying papain, we observed that the yield of mature papain
recovered following in vitro trans-activation was much lower
for mutants at position 175 than for the wild-type enzyme. Using
Western blot analysis, we have shown previously that the total amount
of propapain produced was not affected by the mutations(22) .
Therefore, the differences in yield are not consequences of variations
in the transcription or translation efficiency or intracellular
instability of the proteins. The reduction in yield, which is more
pronounced for the Asn
Ala mutant, could, however,
reflect an increased susceptibility to proteolytic degradation by
subtilisin in the activation step for the mutants. This suggests that
some of the molecules may not be properly folded. Since unfolded
proteins are often found to aggregate(26) , we have measured the
solubility of propapain mutants and the sensitivity of soluble and
insoluble fractions to degradation by subtilisin. In the presence of
exogenous proteases, the 38-kDa wild-type propapain is fully converted
into 24-kDa mature papain (23). This limited proteolytic processing can
be easily distinguished from more extensive and less specific
degradation of unfolded mutants. A large proportion of propapain
mutants at position 175 is found in the pellet fraction (Fig. 2A, lanes P-), the effect being
more pronounced for the Asn
Ala mutant. The pellet
fraction is completely degraded by subtilisin (Fig. 2A, lanes P+), whereas the soluble fraction (Fig. 2A, lanes S-) is fully converted to
mature papain (Fig. 2A, lanes S+). The
ratio of aggregated to soluble fraction is about 0.3 for the wild-type
propapain (Fig. 2B). This ratio is close to 30 and about
50 for the Asn
Gln and Asn
Ala mutants, respectively. The Cys
Ser mutant was
used as a control and has a behavior similar to that of wild-type
propapain (Fig. 2, A and B). Thus, the
partitioning of propapain between soluble, protease-resistant fractions
and insoluble, protease-susceptible fractions is markedly altered by
the replacement of Asn
.
The previous results suggest
that mutation at position 175 has a detrimental effect upon the ability
of the proenzyme to fold in the cell but that, when the protein is
folded, it becomes resistant to proteolytic degradation. However,
subtilisin can selectively remove the pro region of the precursor and
release mature active papain. To determine if mutations at position 175
affect the stability of mature papain, we have measured the rate of
thermal inactivation of papain mutants as defined
previously(22) . The mature enzyme is known to be highly stable
to thermal inactivation, as shown by the half-life of 12.5 ± 0.9
min measured for wild-type papain at 82 °C. For the Asn
Gln and Asn
Ala mutants, the
half-life times at 82 °C are 7.3 ± 0.6 min and 4.6 ±
0.3 min, respectively (Fig. 3), indicating that the mutations
also have an effect on the thermal stability of the mature enzyme.
), Asn
Gln (
), and Asn
Ala (
)
mature papain were incubated at 82 °C for various periods of time
under conditions described under ``Experimental Procedures,''
and the level of remaining activity was measured. Each point is the
average value of three (Asn
mutants) or two (wild-type)
independent measurements.
Kinetic Characterization
The papain mutants
Asn
Gln and Asn
Ala used for
kinetic characterization were purified by covalent affinity
chromatography. The kinetic parameters at optimum pH (6.5) for
hydrolysis of Cbz-Phe-Arg-MCA by the Asn
mutants and
wild-type papain are given in Table I. Removal of the Asn
side chain by replacing asparagine by an alanine residue leads to
a marked 150-fold decrease in (k
/K
)
at pH 6.5. This effect on activity can be entirely attributed to
a decrease in k
, which is 0.38 s
for the mutant Asn
Ala as compared to 41.6
s
for wild-type papain. Mutation of residue 175 to
an alanine therefore has a marked effect on the activity of papain.
However, if the Asn
residue is replaced by a glutamine,
the kinetic parameters for the mutant show relatively little deviation
from those of wild-type. The (k
/K
)
value for Asn
Gln is 135
10
M
s
, a value only
3.4-fold lower than that of wild-type enzyme. Replacing an Asn by a Gln
can be considered as an insertion of an extra methylene group in the
side chain of the Asn residue, and the enzyme seems to be able to
tolerate this modification as determined by the kinetic properties of
the enzyme.
/K
)
for the Asn
Gln and Asn
Ala mutants is illustrated in Fig. 4(A and B,
respectively). The pH activity profiles of the mutants are
significantly narrower than that of wild-type papain (represented by a dashedline in the figures), particularly in the case
of the Asn
Ala mutant. Once again, replacement of
Asn
by an alanine has a more pronounced effect than
mutation to a glutamine. The pH activity profiles of the mutant enzymes
can be fitted to an equation describing a model where two
pK
values (i.e. two ionizable
groups) are considered, one for each limb of the bell-shaped profile.
For the wild-type enzyme, the profile is best described by a
three-pK
model, the additional ionizable
group influencing the activity of the enzyme only in the low pH
region(21) . Due to the precision of our experimental
measurements with the mutant enzymes, we cannot rule out the
possibility that a third ionizable group also modulates the activity in
the acid limb of the pH activity profiles for the mutants. However,
this group would have only a small effect on activity, as observed with
wild-type enzyme(21) . In addition, since the low pH limb of the
profile for the mutant enzymes is displaced to higher pH values, the
third ionizable group might not modulate the activity in the pH range
where the Asn
Ala and Asn
Gln
mutants are active. The value of pK
, which is
4.54 for wild-type papain (see Table I) increases to 5.42 for
Asn
Ala. Similarly, pK
is seen to decrease significantly from 8.45 in wild-type papain
to 7.75 in the Asn
Ala variant. These variations
in the pK
values of the ionizable groups
that modulate the activity of papain are the largest observed so far
with mutants of this enzyme.
/K
)
for the Asn
Gln (panel A) and Asn
Ala (panel B) mutants. The solidline represents the best fit to Reaction 1, obtained by
nonlinear regression of the data to Equation 1. The corresponding pH
activity profile for wild-type papain is included for comparison (dashedline).
Considerations on the Stability of the
Thiolate-Imidazolium Form of Papain
It is generally accepted
that the active form of papain consists of a thiolate-imidazolium ion
pair(9, 10, 11, 12) . The stability of
this ion pair is considered to be very sensitive to its environment. In
the present study, a perturbation of the ion pair is a likely
possibility since Asn interacts directly with one of its
members. The O
atom of Asn
is
hydrogen-bonded to N
of His
, and this
interaction could be important for stabilization of the
thiolate-imidazolium ion pair form of the active site residues in
cysteine proteases. It has been shown previously that any factor
influencing the ion pair stability will consequently have an effect on
the observed activity
((k
/K
)
)
of the enzyme(27) . In the same study, it was also shown that
using certain assumptions, the effect of a mutation on ion pair
stability and on the intrinsic activity (k
/K
) of papain
can be dissected out by a detailed analysis of the pH activity profile.
, His
, or Asn
). To be
applicable to the analysis of mutations at position 175 of papain, the
equations deduced from the model need to be expanded. In its simplest
form, the model describing the ionization pathways of the active site
residues is represented in Fig. 5. The four protonation states of
the active site residues are considered, and K
is
an equilibrium constant used to describe the conversion of the neutral
form (-SH, -Im) to the ion pair form
(-S
, -ImH
) of these
residues. In the previous study(27) , equations were derived
assuming that the difference between pK
and
pK
, the intrinsic pK
values for the ionization of Cys
and
His
in absence of factors stabilizing the ion pair, is
the same for both wild-type and mutant enzymes. In the present study,
this condition is not necessarily met and a more general equation has
to be introduced (Equation
2),
and His
of
papain and cysteine proteases in general.
where pK = (pK
- pK
)
-
(pK
-
pK
)
, the variation in width of
the pH activity profile on going from wild-type papain to the mutant
enzyme;
pK
= (pK
- pK
) and
pK
= (pK
-
pK
) are the variations in the intrinsic
pK
values, pK
and
pK
, resulting from the mutation; K
and K
represent the
equilibrium constant K
for the mutant and
wild-type enzymes, respectively. Since the value of
(
pK
-
pK
)
could be non-negligible, it may partially mask or amplify the effect of
a variation in K
on the width of a pH activity
profile. In Fig. 6A, the variation of
pK with ion pair stability (i.e. -log(K
/K
)) is
simulated for values of (
pK
-
pK
) ranging from -1 to
+1. It is evident from Fig. 6A that the value of K
determined from
pK is
strongly dependent on (
pK
-
pK
). It can be seen also that the narrowing
of the pH activity profile reaches a maximum when the ion pair is
destabilized approximately 100-fold or more (i.e. -log(K
/K
)
2). Further reduction in ion pair stability does not lead to
additional narrowing of the profile, and when the pH activity profile
of a mutant enzyme reaches this theoretical maximum value of
pK, it is only possible to put a higher limit to the
value of K
. However, the relationship between (k
/K
)
and K
is linear when the ion pair is
significantly destabilized (Fig. 6B), indicating that
perturbation of the ion pair will contribute to a decrease in (k
/K
)
even past the limit where no further effect on pH activity
profiles is discernible. Therefore, caution has to be used when
interpreting results of pH activity measurements in terms of
perturbation of the stability of the ion pair form of the active site
residues.
pK) and the perturbation of ion
pair stability for various values of (
pK
-
pK
). Panel B,
relationship between the variation in the experimentally determined
activity (i.e. limiting k
/K value obtained from pH activity profiles) and the perturbation of
ion pair stability.
It is important to dissect out the contribution of ion
pair stability to the measured kinetic parameters when trying to
elucidate the role of Asn in the catalytic mechanism of
papain. From the above considerations, it is obvious that this cannot
be accomplished in a straightforward manner to yield a definitive
answer. However, the data can be interpreted to define limits to the
contribution of Asn
to various aspects of the catalytic
mechanism. With the Asn
Gln mutant, the hydrogen
bond between the side chains of residues 175 and 159 is believed to be
maintained (see below), and in a first approximation we can consider
that (
pK
-
pK
) = 0 for this mutant. Since
pK = -1.07 for Asn
Gln (Table I), we can calculate that K
= 0.58 and replacement of Asn
by a glutamine
causes a 7.6-fold destabilization of the ion pair form compared to
wild-type papain. Knowing the effect of the mutation on the ion pair
stability, we can now calculate the effect on the intrinsic activity of
the enzyme. To do this, we can use the previously determined equation
(Equation 3),
/K
for
mutant over wild-type papain), (k
/K
)
is the intrinsic value of k
/K
for wild-type
papain, and (K
/(K
+ 1)) reflects how the perturbation of K
resulting from a mutation will affect the measured specificity
constant(27) . For Asn
Gln, f = 0.67 (Table II), indicating that the intrinsic
activity of the mutant is only 1.5-fold lower than that of wild-type
papain.
pK
-
pK
) might not be negligible for
the Asn
Gln mutant, limits can be put on the
contribution of Asn
to the catalytic mechanism by
considering that ion pair destabilization is responsible for none or
all of the observed variation in activity. In the first case where the
stability of the ion pair is considered not to be affected by the
mutation, K
= 4.4 and the decrease in
activity is due in totality to a decrease in intrinsic activity of the
enzyme. With
pK = -1.07, we can
calculate (
pK
-
pK
) and f using Equations 2 and 3,
respectively. In the second limiting case, we consider that all of the
effect on the experimentally determined specificity constant originates
from a perturbation of the ion pair and that mutation of Asn
to Gln has no influence on the intrinsic activity of the enzyme, i.e.f = 1. The results using both
assumptions, given in Table II, place reasonable limits on the
magnitude of the effect that mutation of Asn
to a
glutamine can have on the intrinsic activity and on the ion pair
stability of papain (assuming of course that the Asn
Gln mutation does not stabilize the thiolate-imidazolium
ion pair or increase the intrinsic activity of the enzyme). It can be
seen in Table IIthat the conclusions are similar to when the
variation in (
pK
-
pK
) was considered negligible, i.e. the mutation has only a small effect on ion pair stability and/or
intrinsic activity.
Ala mutant,
only the two limiting cases were considered since the probability that
the value of (
pK
-
pK
) is affected by the mutation is much
higher. In the first limiting case (no effect on ion pair stability),
the value of f = 0.0072 indicates that the intrinsic
activity of the mutant Asn
Ala is lower than that
of papain by a factor of no more than 140. By considering that the
mutation has no effect on intrinsic activity (case 2), K
= 0.0060, a value 735 times lower than
that of wild-type papain, which places an upper limit to the ion pair
destabilization upon mutation of Asn
to Ala.
in papain with an alanine
or a glutamine residue by site-directed mutagenesis. The kinetic data
obtained with the substrate Cbz-Phe-Arg-MCA indicate that Asn
can be replaced by a Gln residue without major changes in the
specificity constant (k
/K
)
,
while mutation to an Ala residue leads to a 150-fold decrease in
activity. The side chain of a glutamine retains the possibility of
forming a hydrogen bond with the side chain of His
and
the higher activity of the Asn
Gln mutant compared
to Asn
Ala could be explained by the existence of
such a hydrogen bond. Computer modeling indeed suggests that the
hydrogen bond distances between the side chain amide of residue 175 and
His
in wild-type papain can be maintained in the
Asn
Gln mutant. When the possibility of residue
175 forming such a hydrogen bond to His
is removed, i.e. by mutating Asn
to an alanine, the
catalytic efficiency is reduced by about 2 orders of magnitude.
However, the Asn
Ala mutant still hydrolyzes the
substrate Cbz-Phe-Arg-MCA at a rate much higher than the non-catalytic
rate; therefore, Asn
cannot be considered as an essential
catalytic residue in the cysteine protease papain.
and His
residues as an ion pair at neutral pH, and from theoretical
considerations Asn
has been proposed to stabilize the
thiolate-imidazolium ion pair at the active site of papain(19) .
For a linear peptide containing non-interacting cysteine and histidine
residues, if K
is used to designate the ratio of
the concentration of the peptide where both side chains are ionized to
the concentration where both side chains are neutral, then it can be
shown that log(K
) = (pK
His - pK
Cys). By using
9.1 and 6.4 for the pK
values of cysteine
and histidine, respectively(28) , we obtain a value of 0.0020
for K
. The value of the corresponding equilibrium
constant in papain between the ion pair form and the neutral form of
the active site residues has been estimated at 4.4(27) .
Therefore, in wild-type papain the ion pair is approximately 2200-fold
more stable than if the Cys and His residues were non-interacting in a
linear peptide (K
/K
in Table II). The mutation of Asn
to Gln is accompanied
by an 8-fold decrease in the stability of the thiolate-imidazolium ion
pair. Once the ion pair is formed, there is virtually no difference in
activity between Asn
Gln and wild-type papain (f = 0.67), suggesting that the advantage of having an
Asn at position 175 over a Gln is mainly to stabilize the ion pair. If
limiting cases are considered, the value of K
can
decrease by up to 13-fold while the effect on intrinsic activity is of
no more than 3-fold (f = 0.30). For the Asn
Ala mutant, a major perturbation (narrowing) of the pH
activity profile is observed and the kinetic data can only be used to
put limits to the magnitude of the effects on intrinsic activity and
ion pair stability. For example, if the replacement of Asn
by an alanine has a negligible effect on the intrinsic activity
of the enzyme (case 2 in Table II), the ion pair stability would
be decreased by 735-fold (K
= 0.0060
compared to 4.4 for wild-type papain), to a value of K
that is only 3 times that of K
for
non-interacting residues in a linear peptide. For case 1, where the
decrease in observed activity is suggested to be entirely due to a
decrease in intrinsic activity, the mutation would have no effect on
ion pair stability. It must be noted, however, that in limiting case 1,
a relatively high value of (
pK
-
pK
) = -1.58 is needed to account
for the experimental data. It is most likely that the variations in
kinetic parameters observed for the Asn
Ala
mutation are the result of a combination of effects on ion pair
stability and intrinsic activity, i.e. intermediate between
cases 1 and 2.
Ala mutant. However, it is
interesting to note that, according to the model linking ion pair
stability to pH activity profiles(27) , we would expect a strong
perturbation in ion pair stability to be accompanied by an important
narrowing of the pH activity profile. The quantitative interpretation
of pH activity data depends, however, on the correct assignment of
pK
values to active site groups of the
enzyme. Recently, the possibility that the increase in k
/K
at low pH
shown in Fig. 4could be the result of ionizations other than
that of Cys
in the papain molecule was raised(29) .
If this is the case, the pK
values
measured would not reflect ion pair formation. Changes in the pH
activity profile would be the result of variations in reactivity of
different protonic forms of the enzyme, without variations in the
pK
values of the groups that modulate
activity. This model is relatively complex and requires a large number
of parameters to describe the pH activity profiles. Even though this
possibility cannot be ruled out unequivocally, we believe that ion pair
destabilization leading to narrowing of pH activity profiles is the
most likely explanation for our results. The fact that both the acid
limb and basic limb pK
values are
affected by the mutations provides a strong argument in favor of a
perturbation of ion pair stability. A probable (but not necessary)
consequence of ion pair destabilization is that both the acid and basic
limbs will be affected. Even though the assignment of the ionization of
Cys
to either one of the two pK
values observed in the acid limb of the pH activity profile
for the wild-type enzyme cannot be made unambiguously, as concluded
previously(27) , the data presented for mutants of Asp
by Ménard et al.(27) and for mutants of
Asn
(this paper) can all be rationally explained by
considering electrostatic effects and ion pair perturbation on a
relatively simple model considering in a first approximation only one
active form of the enzyme and three ionizable groups. Although more
complex explanations cannot be ruled out, we continue to favor the
simplest model that fully accounts for the experimental results
presented in this report and all related reports originating from this
laboratory. Experiments are in progress, however, to clarify this
point.
side chain
in papain by that of an Ala residue is evaluated to decrease the
intrinsic activity of the enzyme by a factor of no more than 140. For
serine proteases, mutation of the Asp residue to Ala (for subtilisin)
and Ser (for trypsin) resulted in approximately 10
-fold
reductions in enzymatic activity(31, 32) . However, an
Asp
Asn mutant of trypsin displayed only
10
- to 10
-fold decreases in activity, depending
on the nature of the leaving group of the substrate(33) . This
latter mutation is peculiar in that the side chain can form a hydrogen
bond with the active site His residue(34) . In addition, it has
been shown that even though the presence of the negative charge
adjacent to His
in trypsin is important for activity, its
precise location is not critical. Indeed, an alternate geometry for the
catalytic triad of serine proteases has been proposed(32) . The
latter two results indicate that certain modifications of the catalytic
triad in serine proteases are tolerated.
.
This hydrogen bond allows rotation of the His
side chain
to orient the imidazole group in a proper position to act as a proton
donor to the leaving group of the substrate. The fact that a C-S bond
is weaker than a C-O bond and that the thiolate anion is a very good
leaving group can explain the necessity of such a step in cysteine
proteases. Therefore, the full catalytic power of the triad might be
better exploited in the hydrolysis of non-activated peptide bonds,
whereas the activity of the Asn
mutants of papain was
measured against a small activated peptidyl substrate. For this reason,
the influence of the mutations on activity against protein substrates
might be more important than the measured effects with the substrate
Cbz-Phe-Arg-MCA, but the very low amount of enzyme available from the
expression system precludes such studies. It must be noted also that
preliminary results with cathepsin S (data not shown) show that
mutation of Asn
has a stronger effect on activity than
that observed for papain, indicating that the magnitude of the
Asn
contribution to enzymatic activity might differ from
one cysteine protease to another.
based exclusively upon
enzymatic activity, given the relatively modest effect of amino acid
substitution at position 175. Indeed, our results show that presence of
a Gln at position 175 is almost neutral with respect to the enzyme
activity. However, within a large data base of cysteine protease
sequences, no residue is found other than an asparagine(37) .
The strict conservation of Asn
might therefore be the
consequence of properties in addition to the catalytic activity of the
enzyme. Wild-type propapain accumulates in the yeast cell vacuole (20) mostly as a soluble, protease-resistant species.
Replacement of Asn
by either a Gln or Ala increased the
fraction of insoluble, protease-susceptible propapain, suggesting that
these mutations alter the ability of the protein to fold into a
functional protease precursor. In addition, the mature papain mutants
resulting from the processing of the properly folded proenzymes have an
increased rate of thermal inactivation, indicating that the mutations
affect the thermal stability of the mature enzyme. The acetamide H
atoms of Asn
in wild-type papain interact with the
aromatic rings of Trp
and Trp
, and
perturbation of these interactions in the Asn
Gln
mutant could contribute to the decrease in stability of the enzyme. The
computer modeling experiments indicate that the
Gln
-His
hydrogen bond can be formed with or
without perturbation of the interactions with the Trp residues and,
therefore, cannot unambiguously support or refute this hypothesis. A
similar structural role has been established recently for the catalytic
histidine residue at the active site of phospholipase
A
(38) . Our results indicate that in addition to its
contribution to the catalytic properties of the enzyme, Asn
participates in the folding pathway (39) and in the
thermal stability of the folded protein. The Asn
residue
in cysteine proteases could constitute another example of the
conservation of an active site residue resulting from a combination of
functional and structural constraints.
Table: Kinetic parameters for hydrolysis of
Cbz-Phe-Arg-MCA by papain variants
Table: Contribution
of the Asn mutations to ion pair stability and
intrinsic activity
©1995 by The American Society for Biochemistry and Molecular Biology, Inc.