Download N-terminal portion acts as an initiator of the inactivation of pepsin at

Protein Engineering vol.14 no.9 pp.669–674, 2001
N-terminal portion acts as an initiator of the inactivation of
pepsin at neutral pH
Takuji Tanaka and Rickey Y.Yada1
Department of Food Science, University of Guelph, Guelph,
Ontario N1G 2W1, Canada
1To
whom correspondence should be addressed.
E-mail: [email protected]
Porcine pepsin, an aspartic protease, is unstable at neutral
pHs where it rapidly loses activity, however, its zymogen,
pepsinogen, is stable at neutral pHs. The difference between
the two is the presence of the prosegment in pepsinogen.
In this study, possible factors responsible for instability
were investigated and included: (i) the distribution of
positively charged residues on the surface, (ii) an insertion
of a peptide in the C-terminal domain and (iii) the dissociation of the N-terminal fragment of pepsin. Mutations to
change the number and the distribution of positive charges
on the surface had a minor effect on stability. No effect on
stability was observed for the deletion of a peptide from
the C-terminal domain. However, mutations on the Nterminal fragment had a major impact on stability. At pH
7.0, the N-fragment mutant was inactivated 5.8 times slower
than the wild-type. The introduction of a disulfide bond
between the N-terminal fragment and the enzyme body
prevented the enzyme from denaturing. The above results
showed that the inactivation of pepsin was initiated by the
dissociation of the N-fragment and that the sequence of
this portion was a major determinant for enzyme stability.
Through this study, we have created porcine pepsin with
increased pH stability at neutral pHs.
Keywords: enzyme stability/N-terminal fragment/pepsin/pepsinogen/pH
Introduction
Pepsin (EC 3.4.23.1) belongs to the aspartic protease family
and functions under highly acidic conditions. Enzymes in the
aspartic protease family are believed to be ubiquitous and
share various similarities in such properties as conformations
(Wlodawer et al., 1985; Danley et al., 1989; McKeever et al.,
1989; Gilliland et al., 1990; Jaskólski et al., 1990; Newman
et al., 1991, 1993; Chen et al., 1992; Tong, et al. 1993; Yang
et al., 1997; Lee et al., 1998) and catalytic mechanism (Khan
and James, 1998; Richter et al., 1998). Despite the similarities
which define the aspartic proteinase family, differences in
functionality, e.g. substrate specificities, do exist; thus the
aspartic proteinases are ideal models to demonstrate structure–
function relationships of enzymes.
All proteins are synthesized in a neutral pH environment,
thus their natural conformational state and functionality exists
in this environment. However, most aspartic proteinases are
stable under acidic conditions and become irreversibly denatured under neutral pH conditions (Bohak, 1969; Fruton, 1971).
In the case of pepsin, its zymogen, pepsinogen, is stable under
neutral pH conditions. Since the majority of the pepsin
© Oxford University Press
molecule is identical to pepsinogen, minor differences may be
responsible for pepsin’s instability at neutral pHs.
The most obvious differences between pepsin and pepsinogen are found in the prosegment portion. Not only does
the prosegment cover the active site cleft, but it contains a
large number of positively charged residues, i.e. 13 (nine Lys,
two Arg, two His) out of the 44 residues in the prosegment
of pepsinogen are positively charged (James and Sielecki,
1986; Lin et al., 1989). Pepsin on the other hand contains
only four positive residues (two Arg, one Lys, one His).
Therefore, it is evident that the number and distribution of
these positively charged residues contribute to the difference
in pH stability between pepsin and pepsinogen. In contrast,
unlike most aspartic proteinases, chicken pepsin is relatively
stable at neutral pHs (Bohak, 1969). A homology search
between porcine and chicken pepsin shows a 74% similarity.
Major differences between chicken and porcine pepsins are in
charge distribution and the N-terminal amino acid sequences.
In chicken pepsin, there are 21 Asp, 13 Glu, 9 Lys, 5 Arg and
4 His charged residues, while in porcine pepsin, 28, 13, 1, 2
and 1 are charged, respectively. For both porcine and chicken
pepsin, most of these charged residues are distributed on the
surface; however, the distribution of the positively charged
residues is more favourable in chicken pepsin.
Another difference between pepsin and pepsinogen is the
position of the N-terminal fragment. In a zymogenic form and
during activation, the N-terminal fragment is in the active site
cleft. After activation, this N-terminal fragment is placed in
the β-sheet at the bottom of the protein. This relocation results
in about a 40 Å movement (James and Sielecki, 1986). Since
this portion relocates from one side of the protein to other
side, it is suggested that this fragment could be readily moved
from its position and could be important for the stabilization
of pepsin under neutral pH conditions. Lin et al. reported
denaturation of pepsin begins with the denaturation of the Nterminal domain, and suggested that the interaction between
the N-fragment and the enzyme body is important for stability
(Lin et al., 1993). A recent crystallographic study of cathepsin
D, another neutral pH stable aspartic proteinase, showed that
the N-terminal fragment of this enzyme is relocated to its
active site cleft at pH 7.5, resembling its zymogenic form (Lee
et al., 1998).
From the above observations, several key factors in the
instability of pepsin are suggested. In this study, the following
were undertaken in order to investigate and possibly improve
the stability of pepsin at neutral pHs: (i) the alteration of the
charge distribution on the surface of pepsin, (ii) the deletion
of the extra amino acid sequence observed in porcine pepsin
C-domain, (iii) the addition of hydrogen bond stabilizers and
(iv) the stabilization of the N-terminal portion of pepsin
through mutations and the introduction of a disulfide bond.
Materials and methods
Materials
The expression plasmid pTFP1000, which contains the gene
for wild-type porcine pepsinogen fused to the 3⬘ end of the
669
T.Tanaka and R.Y.Yada
gene for thioredoxin, was constructed according to Tanaka and
Yada (Tanaka and Yada, 1996). Restriction endonuclease
NheI and DNA-modifying enzymes were purchased from Life
Technologies (Gaithersburg, MD). Restriction endonucleases
XbaI and BglII were from Boehringer Mannheim (Laval, PQ,
Canada) and New England Biolabs (Mississauga, ON, Canada),
respectively. Oligonucleotide primers used for mutagenesis
and the synthetic octapeptide KPAEFF(NO2)AL (ss 1) were
synthesized at the Institute of Molecular Biology and Biotechnology at McMaster University (Hamilton, ON, Canada). The
synthetic oligopeptide LSF(NO2)NleAL methyl ester (ss 2)
was obtained from Sigma Chemical (St Louis, MO). Casamino
acids were purchased from Difco (Detroit, MI). DEAE
Sepharose CL-6B was from Pharmacia Biotech (Uppsala,
Sweden). All other reagents were of the purest grade commercially available.
Mutagenesis of pepsinogen
Site-directed mutagenesis was carried out by the method of
Zoller and Smith (Zoller and Smith, 1982) using a uracilcontaining single-stranded M13mp19 DNA template (Kunkel,
1985). The following oligonucleotide primers were used:
G2C: 5⬘-CCG CTG CCC TCA TAT GCG ATG AGC C-3⬘;
G2S/D3Y: 5⬘-GCCCTGATATCATATGAGCCC-3⬘;
L10M/T12A/E13S: 5⬘-GAACTACATGGATGCATCGTACTTTGGC-3⬘;
S46K: 5⬘-GTCTACTGCAAAAGCTTAGCCTGCAGC-3⬘;
D52N/N54K/Q55R: 5⬘-CCTGCAGCAACCACAAGCGCTTCAACCC-3⬘;
D60K: 5⬘-CAACCCTGATAAATCGTCGACCTTCGAG-3⬘;
L167C: 5⬘-CGATGCCGCCGCACAGTACTACACTGCCGC-3⬘;
S196R: 5⬘-CAGATTACCCTCGATCGCATCACC-3⬘;
D200G/E202K: 5⬘-CACCATGGCCGGCAAGACCATCG-3⬘;
∆240–246/⫹GD: 5⬘-GGAGCCAGCGAGGGCGATATCAGCTGCTCC-3⬘;
S254K: 5⬘-CTGCTCCTCAATTGACAAGCTGCCTGAC-3⬘.
Some mutations were also incorporated with other mutations.
The following combinations of mutations were constructed:
G2C/L167C;
G2S/D3Y [N-frag(A)];
G2S/D3Y/L10M/T12A/E13S (N-frag);
L10M/T12A/E13S [N-frag(B)];
S46K/D52N/N54K/Q55R/D60K (N-DOM);
S196R/D200G/E202K (C-DOM);
S46K/D52N/N54K/Q55R/D60K/S196R/D200G/E202K (N⫹C);
∆240–246/⫹GD (Del).
G2C/L167C, N-frag(A), N-frag and N-frag(B) were intended to
stabilize the N-terminal portion of pepsin. N-DOM, C-DOM
and N⫹C were made to alter the charge distribution. Del
was undertaken to remove the putative mobile portion in the
C-terminal domain.
Expression of Trx-PG
Mutant pepsinogen DNA was cloned into the expression plasmid
pTFP1000, which contains the gene for wild-type pepsinogen,
by replacing the corresponding fragment of mutant pepsinogen
DNA using restriction enzymes. The resultant plasmids were
used to transform Escherichia coli GI724 by the method of
Hanahan (Hanahan, 1983). Escherichia coli GI724 cells carrying
the plasmid were cultured in 1.5 l of induction media (1⫻ M9
salts, 0.2% casamino acids, 0.5% glucose, 1 mM magnesium
670
chloride) containing 0.15 mg/ml ampicillin at 30°C. When the
culture reached early log phase (absorbance at 550 nm ⫽ 0.5),
expression of Trx-PG was induced through the addition of 15
ml of a 10 mg/ml L-tryptophan solution. The culture was
incubated for a further 6 h, after which the cells were harvested
by centrifugation at 8000 g for 10 min at 4°C.
Isolation and purification of Trx-PG
Expressed Trx-PG was extracted from the cells by sonication
and purified through ammonium sulfate precipitation followed
by chromatography on anion-exchange columns DEAE-Sephacel
and DEAE-Sepharose CL-6B. Purified Trx-PG was dialysed
against 20 mM Tris–HCl buffer pH 7.5, for 2 h at 4°C, filter
sterilized and stored at 4°C. Purity was assessed by SDS–PAGE,
according to Laemmli (Laemmli, 1970), followed by staining
with Coomassie Blue.
Kinetics
Preparation of pepsin for each mutant Trx-PG and determination
of the kinetic parameters were as previously described (Tanaka
and Yada, 1996). Pepsin was obtained from Trx-PG by acidification and purification through gel filtration. Kinetics were
measured using peptide ss 1 and ss 2 at a substrate concentration
range of 0.01–0.2 mM at 37°C. pH employed were pH 2.1 for
ss 1 and pH 3.95 for ss 2. The protein concentration was
determined as the amount of pepstatin required to completely
inhibit pepsin.
Stability tests
Recombinant pepsinogen was activated and the corresponding
pepsin was purified through Sephadex G-50 gel filtration with
20 mM sodium acetate buffer (pH 5.3). An aliquot of 40 µl of
this preparation (0.1 mg/ml protein) was mixed with 40 µl of
dH2O and 80 µl of 0.4 M buffer (sodium phosphate buffer pH
7.0, 7.5, 8.0). Small aliquots were withdrawn at specific times
and quenched with 3 M sodium acetate buffer (pH 5.2). Residual
activity was determined with the synthetic peptide substrate
(ss 1) at pH 2.1, 25°C.
To investigate the effects of additives, the same stability test
was conducted; however, a 40% (v/v) glycerol/40% (w/v) sucrose
solution was used to replace the dH2O in the above test.
Disulfide bonds do not spontaneously form under pH 6.0,
thus to accelerate the formation of the disulfide bond in the
G2C/L167C mutant, several oxidizing reagents were tested. The
pepsin preparation was incubated in 0.1 M Tris–malate buffer
(pH 4.0, 5.0, 6.0, 6.6, 7.2) with an oxidizing reagent, i.e. 5 mM
K3Fe(CN)6, 5 mM FeCl3, 1 mM o-iodothobenzoate, 0.5 mM
dithionitrobenzoate or 0.5 mM dithiopyridine, for 24 h at room
temperature (or 4°C). After oxidization, each sample was tested
for stability.
Results and discussion
Kinetics of activated pepsin
No mutations studied in this work were made at or near the
active site. It was anticipated that the mutations conducted in
this study would not affect enzyme activity although our previous
study on the sole lysine residue on pepsin showed a single
mutation outside of the active site could change enzyme activity
(Cottrell et al., 1995). Reaction kinetics were, therefore, determined for two synthetic substrates, KPAEFF(NO2)AL and
LSF(NO2)NleAL (Table I). Near the optimum pH of pepsin (pH
2.1), mutants had k0/Km values of 0.52–2.88 while wild-type
had a k0/Km value of 1.92. The activities of mutants were very
high, thus the structures were likely to be affected at small
N-terminal fragment and neutral pH inactivation of pepsin
Table I. Kinetic constants of mutant and wild-type pepsin
Enzyme
ss 1 (KPAEFF(NO2)AL)
Km (µM)
Wild-type
N⫹C
DEL
N-frag
N-frag(A)
N-frag(B)
G2C/L167C
34
57
70
83
75
65
80
⫾
⫾
⫾
⫾
⫾
⫾
⫾
5
5
6
15
8
9
7
ss 2 (LSF(NO2)NleAL methyl ester)
k0 (/s)
k0/Km (/s/µM)
Km (µM)
65.4 ⫾ 3.1
77.5 ⫾ 2.9
60.9 ⫾ 2.6
68.9 ⫾ 6.6
186.0 ⫾ 9.8
187.1 ⫾ 12.8
41.6 ⫾ 2.0
1.92 ⫾ 0.30
1.36 ⫾ 0.13
0.87 ⫾ 0.08
0.83 ⫾ 0.17
2.48 ⫾ 0.30
2.88 ⫾ 0.44
0.52 ⫾ 0.03
19
31
15
28
28
41
18
⫾
⫾
⫾
⫾
⫾
⫾
⫾
6
13
2
7
7
5
1
k0 (/s)
k0/Km (/s/µM)
190.5 ⫾ 18.9
50.7 ⫾ 9.4
25.7 ⫾ 1.0
80.7 ⫾ 10.2
122.0 ⫾ 15.6
315.3 ⫾ 16.8
13.0 ⫾ 0.22
10.0
1.64
1.71
2.88
4.36
7.69
0.72
⫾ 3.3
⫾ 0.75
⫾ 0.24
⫾ 0.81
⫾ 1.22
⫾1.02
⫾ 0.17
Values represents the mean of three determinations ⫾ SD. Reactions were carried out at pH 2.1 for ss 1 and pH 3.95 for ss 2, both at 37°C.
Table II. Rate constants of inactivation of mutants at pH 7.0
Enzyme
kd (/min)
N-frag
N-frag (glycerol, sucrose)
N-frag (pH 7.5)
G2S/D3Y [N-frag(A)]
L10M/T12A/E13S [N-frag(B)]
G2C/L167C
N⫹C
N-DOM
C-DOM
DEL
Wild-type
Wild-type (glycerol)
Wild-type (sucrose)
Wild-type (glycerol, sucrose)
Wild-type (pH 7.5)
0.0169 ⫾ 0.0014
0.00229 ⫾ 0.00031
0.268 ⫾ 0.008
0.0680 ⫾ 0.0038
0.0646 ⫾ 0.0015
0.0536 ⫾ 0.0051
0.0421 ⫾ 0.0071
0.0905 ⫾ 0.0173
0.0852 ⫾ 0.0237
0.0851 ⫾ 0.0088
0.0991 ⫾ 0.0075
0.0365 ⫾ 0.0026
0.0203 ⫾ 0.0009
0.00743 ⫾ 0.00216
ND (too rapid)
ND, inactivation was too rapid (completed within 5 min) under the
experimental conditions to determine the inactivation rate. Values represent the
means of three determinations ⫾ SD.
degrees. At a higher pH (pH 3.95), all the mutants showed
lower activities, although the activities were high if their low
Km values are considered.
Stability and additives
The time course of inactivation of wild-type and mutant pepsin
is shown in Figure 1. The inactivation rate constants were
calculated from the plots and summarized in Table II.
The inactivation of wild-type pepsin was measured at pH 7.0
and 7.5. At pH 7.0, pepsin was rapidly inactivated at a rate of
0.099/min (open circles in Figure 1A). At this rate, virtually all
activity ceased within 1 h. At pH 7.5, pepsin was rapidly
inactivated and lost activity in 5 min (open circles in Figure 2B).
Lin et al. suggested that inactivation was caused by irreversible
denaturation which was triggered by the denaturation of the
N-terminal domain (Lin et al., 1993). Therefore, it may be
possible to stabilize the protein by preventing this denaturation.
In a previous study, it was found that glycerol was able to
preserve unstable mutants of fused pepsinogen (Richter et al.,
1999). Thus a hydroxyl group rich compound may aid in
reducing denaturation. In the present study, glycerol and sucrose,
another hydroxyl group rich compound, were chosen. Both
additives had no effect on the activity of wild-type under the
experimental conditions employed.
At 20% (w/v), both sucrose and glycerol slowed inactivation
(Figure 1A). The rate of inactivation (Table II) was reduced to
0.0203 and 0.0365/min at pH 7.0, respectively. Ten to 30% of
the initial activity was retained after 60 min at pH 7.0 using
Fig. 1. Inactivation of wild-type and mutant pepsin. Pepsin was inactivated in
the conditions described in the text and residual activities were plotted.
(A) Open circle, wild-type; closed square, wild-type with glycerol; open
triangle, wild-type with glycerol and sucrose. (B) Closed circle, wild-type;
open triangle, N⫹C; closed triangle, N-fragment; open circle, ∆240–246; open
square, N-frag(A); closed square, N-frag(B). Each data point represents the
mean of three determinations.
these additives. Moreover, the combination of 20% glycerol and
sucrose reduced the inactivation rate to 0.00743/min and resulted
in half of the initial activity after 60 min.
Denaturation can result from either the rearrangement of the
hydrogen bonds and/or electrostatic interactions. Since denaturation is triggered by a rise in pH, electrostatic interactions are
most likely to be involved. Although electrostatic interactions
are not affected by hydroxyl groups, the addition of glycerol/
sucrose did help stabilize pepsin. At the same time, the addition
of glycerol/sucrose did not result in 100% stabilization since
some pepsin activity was still lost; therefore, both electrostatic
interactions and hydrogen bonds are believed to contribute to
the denaturation of pepsin.
Stability of the mutants (charge distributions)
Change of the distribution of negatively charged residues on
the surface
Pepsin has only a few positively charged residues but an
abundance of negatively charged residues on its surface. Given
that pepsin is an acidic protein, it is reasonable to expect that
671
T.Tanaka and R.Y.Yada
Fig. 3. Comparison of the sequence of the N-terminal fragment. From the
comparison of porcine and chicken pepsin sequences, three mutants were
planned. Chicken pepsin has three extra amino acids on the N-terminal end.
The addition of these amino acids to porcine was not considered because it
could interfere with the activation itself. The substitution amino acid residues
in the mutants are indicated using underlining.
Fig. 2. Inactivation of the N-terminal fragment mutant. Inactivation test of
N-fragment, N-frag(A) and N-frag(B) mutants showed that individual
mutations in the N-fragment mutant were not critical for the stabilization of
pepsin. Both N-frag (A) and (B) mutants showed slight stabilization effects
(B) and were similar in stability (A) to the wild-type while the N-frag mutant
showed drastic stabilization. (A) The inactivation reactions were carried out at
pH 7.0. (B) The inactivation reactions were carried out at pH 7.5. Symbols
are: wild-type (open circle); N-frag(A) (inverted open triangle); N-frag(B)
(open square); N-fragment (upright open triangle); wild-type with glycerol and
sucrose (closed circle); N-frag(A) with glycerol and sucrose (inverted closed
triangle); N-frag(B) with glycerol and sucrose (closed square); N-fragment
with glycerol and sucrose (upright closed triangle). Each data point represents
the mean of three determinations.
many negatively charged residues occur on the surface. As
pH increases, these residues become deprotonated resulting in
electrostatic repulsion which may lead to instability. Thus
replacement of these negatively charged residues with neutral
or positive residues may stabilize pepsin at neutral pH values.
The results of the mutation
Replacements of the positive residues were chosen in the region
where negatively charged residues were highly concentrated.
Mutations were selected in order to resemble the sequence of
chicken pepsin. Kinetic measurements at pH 2.1 and 3.95 were
taken for the resultant mutant, N⫹C. The mutant showed
comparable kinetic constants to the wild-type; therefore,
mutations on the surface had little effect on the kinetic parameters
(Table I).
Although the N⫹C mutant had similar activity as wild-type,
the stability at neutral pH changed (Table II; Figure 1B). The
mutant inactivated at about half the rate of the wild-type. The
inactivation of the mutant, which had mutations on only the Nor C-terminal domains (N-DOM and C-DOM, respectively), did
not show the same level of stabilization. The rate of the
inactivation of N-DOM and C-DOM was comparable to that of
the wild-type. The results of the above three mutants demonstrated that the distribution of the negatively charged residues
on the surface of the porcine pepsin helped to stabilize the
enzyme, however, the degree of stabilization was not substantial.
Since Lin et al. had shown that the initial denaturation occurred
in the N-terminal domain (Lin et al., 1993), the mutations on
the N-terminal domain were expected to have a dominant effect.
672
The negatively charged residues on the N-terminal domain
themselves, however, only had a minor effect on the stability.
Thus the distribution of the charges over the entire surface must
be changed in order to achieve noticeable stabilization of pepsin
at neutral pH.
Stability of the mutants (N-terminal fragment)
Possible effect of the N-terminal portion on denaturation
The position of the N-terminal fragment in pepsin changes from
the active site cleft to the opposite side of the molecule during
activation (James and Sielecki, 1986). This implies that the Nterminal portion is relatively easily displaced from its original
position. If this N-terminal portion moves away from its original
position, a β-sheet loses one of its strands, raising the possibility
of destabilization of the sheet and/or interaction with another
hydrogen bond source. Therefore, it is suggested that stabilizing
this fragment in its original position, which is a strand of a βsheet on the bottom of pepsin molecule, will prevent denaturation
in the event of neutralization. In order to examine this possibility,
mutations were introduced into the N-terminal portion to keep
it in the fixed position at the bottom of the pepsin molecule.
The comparison of the amino acid sequence of the pepsins
from porcine and chicken (chicken pepsin is relatively stable at
neutral conditions) revealed major differences in the N-terminal
portion. We chose five amino acid residues to be replaced
(Figure 3).
Lin et al. suggested that Asp11 in the N-terminal portion was
critical to the denaturation at neutral pH (Lin et al., 1993).
However, the sequence comparison of pig and chicken pepsins
showed that this position in chicken pepsin was conserved, thus
this residue was not chosen as a mutation site.
The results of the mutation
The mutation of the N-terminal portion was done with two sets
of mutations, G2S/D3Y [N-frag(A)] and L10M/T12A/E13S
[N-frag(B)]. These two mutants and a third mutant which
combined both mutations (N-frag) exhibited similar kinetic
constants to the wild-type and had comparable catalytic activities
for both synthetic substrates (Table I).
Stability tests of the N-frag mutant showed that it was
stabilized by 5.8 times as compared to the wild-type (Table II;
Figure 2A). While the wild-type was inactivated in 60 min, this
mutant retained 30% of its activity after 60 min. Even after 4 h,
1.8% of the original activity was observed with this mutant.
When the pH was raised to 7.5 (Figure 2B), the wild-type
showed no activity after 5 min. Even the addition of glycerol
and sucrose, which was shown to stabilize the enzyme, had no
effect. When the N-frag mutant was exposed to pH 7.5, it
retained 5% activity after 15 min. The rate constant of inactivation
for N-frag at pH 7.5 was 0.268/min. Moreover, in the presence
of both glycerol and sucrose at pH 7.5, the enzyme retained
25% activity after 30 min.
N-terminal fragment and neutral pH inactivation of pepsin
Fig. 4. Molecular model comparison of wild-type and N-terminal fragment
mutant pepsin. Superposition of the N-terminal fragment of wild-type (grey)
and the N-frag mutant (black) models. There were no major differences. Root
mean square deviation between backbone atoms of wild-type and N-frag was
0.28 Å. The numbers show the mutation sites in N-frag.
Since the N-frag mutant had five amino acid replacements,
some of the mutations could be more critical than others. The
G2S/D3Y and L10M/T12A/E13S mutants, however, showed
less stability than the N-frag mutant (Figure 2). Either mutant
showed an inactivation rate which was about 1.5 times slower
than the wild-type while the N-frag mutant was 5.8 times slower.
Also, at pH 7.5, the activities of both N-frag(A) and N-frag(B)
mutants were quenched slower than the wild-type, but faster
than the N-frag mutant. After 5 min, N-frag(A), N-frag(B), Nfrag and wild-type had 0, 3, 27 and 0% of the original activity
in the absence of glycerol and sucrose while 45, 46, 72 and 0%
of the original activities remained in the presence of glycerol
and sucrose, respectively.
Molecular model minimization showed that these mutations
did not contribute to the internal interactions (Figure 4). The
only major difference between the wild-type and N-frag was the
addition of a hydrogen bond between Ser2 γ-oxygen and L167
nitrogen in the G2S mutation. However, kinetics of the N-frag(A)
mutant showed this addition was insufficient to stabilize the
protein entirely. Thus it was concluded that each of the five
replacements by themselves were not critical, but synergistically
helped to stabilize the enzyme.
Since each mutation in itself was not critical to stabilization,
the mechanism of stability was still in question. The authors
suggest two possibilities on how the mutations stabilized pepsin:
(i) the release of the N-terminus portion is prevented with these
mutations or (ii) these mutation sites are responsible for the
stability of the released N-terminal portion. The crystal structure
of inactive cathepsin D at pH 7.5 showed that the N-terminal
portion is relocated into the active site cleft and is stabilized by
an interaction to the catalytic site (Lee et al., 1998). From this
crystal structure data, the authors suspect the second possibility
is more likely.
Disulfide bond formation to fix the N-terminal portion in place
If the mobility of the N-terminal portion initiates denaturation,
then it would be logical to think that fixing this portion to the
enzyme body would prevent denaturation. To fix the N-terminus
portion to the enzyme body, a potential disulfide bond was
introduced. This mutant, G2C/L167C, had a cysteine residue at
the second residue of the N-terminal portion and another cysteine
on the opposite side of the enzyme body (Figure 5). The kinetic
studies of this mutant showed lower, but a substantial amount
of activity as compared to the wild-type (Table I).
The rate constant of the inactivation of G2C/L167C at pH
7.0 was about half of the wild-type but was not as slow as
N-frag. However, inactivation slowed down after 30 min, and
Fig. 5. Molecular model of the introduced disulfide bond in G2C/L167C
mutant pepsin. A molecular model around the mutation site of G2C/L167C is
shown. The introduced disulfide bond, shown by the arrow, is well
accommodated in this position.
Fig. 6. Inactivation of G2C/L167C mutant pepsin. Inactivation of G2C/L167C
mutant and the effect of the oxidizing reagent are shown. At pH 7.5, the
oxidization did not stabilize the wild-type [without oxidization (open square)
and with oxidization (closed square)] while oxidization of G2C/L167C
stabilized the enzyme [without oxidization (open circle) and with oxidization
(closed circle)]. Each data point represents the mean of three determinations.
had a low but noticeable activity (3.2%) over 24 h while the
N-frag, N-frag(A) and N-frag(B) mutants were completely
inactive after 24 h. These results would imply that the formation
of the disulfide bond prevented denaturation by preventing the
movement of the N-terminal portion. However, the slower
disulfide bond formation seemed to compete with the faster
inactivation since substantial activity was lost before reaching
the plateau. To increase the rate of disulfide bond formation,
oxidizing reagents were used, i.e. FeCl3, K3Fe(CN)6, o-iodothobenzoate, dithionitrobenzoate and 3,3⬘-dithiopyridine. These
reagents had comparable effects. After 24 h of oxidization with
2 mM FeCl3, the oxidized G2C/L167C was tested for stability
at pH 7.5 (Figure 6). G2C/L167C, without oxidizers, showed
slower inactivation than wild-type; however, most of the activity
was lost after 480 min. In the presence of FeCl3, inactivation
slowed down and reached a plateau at 20% of its initial activity.
Further inactivation studies showed that activities were retained,
11% at 24 h and 5% at 74 h.
These results indicated that the disulfide bond formation kept
the N-terminal fragment close to its native position, thereby,
stabilizing the enzyme. The formation of disulfide bonds was
faster at pH 7.5 than at pH 7.0, thus a greater stabilizing effect
was observed at the higher pH.
Conclusion
The present study began with the question of which factors
destabilize pepsin at neutral pH. From the differences observed
673
T.Tanaka and R.Y.Yada
among aspartic proteases and between zymogens and activated
enzymes, the authors speculated that both the distribution of the
charges on the surface and the stability of the N-terminal portion
on the enzyme body were vital for stability.
The former possibility was investigated by introducing a
number of positive charges in place of negative charges. The
results indicated that these positive charges resulted in a minor
improvement in stability. Therefore, it is suggested that the
distribution of the charges had an effect only after the denaturation
was triggered by other factors.
The mutations on the N-terminal portion exhibited a larger
contribution to restricting denaturation than the mutations to
alter charge distribution. Replacement of five amino acid residues
in the first 13 residues reduced the inactivation rate by 5.8 times
at pH 7.0. Moreover, in the presence of glycerol and sucrose,
this mutant showed a very low rate of inactivation, 0.00229/
min, and the residual activity after 240 min was 50% of the
initial activity compared to the wild-type which lost most of its
activity in 60 min. This mutation indicated the importance of
the N-terminus to denaturation. Subsequently, the introduction
of a disulfide bond was used to fix the N-terminal portion onto
the enzyme body. Since inactivation was faster than disulfide
bond formation, a large amount of the activity was initially lost,
after which, the rate of inactivation slowed and then plateaued.
These results demonstrated that limiting the N-terminal mobility
limits the denaturation of pepsin at neutral pH.
From the above results, the following was concluded: (i) the
distribution of the charges was important in the stability of
pepsin, (ii) the N-terminal portion was a key segment to
inactivation and (iii) the N-terminal portion was the trigger to
denaturation of the N-terminal domain causing inactivation.
Acknowledgements
The authors are grateful to Ms Erika Tsumura for her technical assistance, Mr
Kenneth Payie and Mark Yoshimasu for their helpful suggestions on preparation
of the manuscript. This research was supported by a grant from the Natural
Sciences and Engineering Research Council of Canada.
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Received February 26, 2001; revised May 9, 2001; accepted June 15, 2001