(Received for publication, July 6, 1995; and in revised form, August 6, 1995)
From the
The amino-terminal propeptide, consisting of 77 amino acid
residues, is known to be required as an intramolecular chaperone to
guide the folding of mature subtilisin E, a serine protease, into
active mature enzyme. Many mutations within the pro-sequence have been
shown to abolish the production of active subtilisin E (Kobayashi, T.,
and Inouye, M.(1992) J. Mol. Biol. 226, 931-933). Here
we report characterization, refolding, and inhibitory abilities of six
single amino acid substitution mutations (Ile
Val, Ile
Thr, Gly
Asp, Lys
Glu, Ala
Thr, and Pro
Leu) and a
nonsense mutation (N59-mer) at the codon for Lys
.
These mutant propeptides were expressed in Escherichia coli using a T7 expression system and were purified to homogeneity.
Surprisingly, Lys
Glu, Ala
Thr and Pro
Leu were found to
still function as a chaperone for in vitro refolding of
denatured subtilisin BPN` with 60, 80, and 54% efficiency compared to
the wild-type propeptide, respectively. The K
values against subtilisin BPN` were 1.6
10
M, 1.2
10
M, and 2.1
10
M,
respectively, almost identical to the K
value exhibited by the wild-type propeptide (1.4
10
M). In contrast, Ile
Val and Gly
Asp were able to
refold denatured subtilisin BPN` with only 18 and 13% efficiencies and
had K
values of 10 and 11
10
M, respectively. The Ile
Thr mutant propeptide was unable to refold denatured
subtilisin BPN` and gave a 100-fold higher K
(118
10
M) than the
wild-type propeptide. The N59-mer propeptide extending from
Leu
to Met
was unable to
function as a chaperone. Like the wild-type propeptide, none of the
mutant propeptides had secondary structures as judged by their circular
dichroism spectra. The present results demonstrate that the ability of
the propeptide as a chaperone to refold the denatured protein is well
correlated with its ability as a competitive inhibitor for the active
enzyme. This supports the notion that the secondary and tertiary
structures of the propeptide are identical or highly homologous between
the renatured propeptide-subtilisin complex and the inhibitory complex
formed between the propeptide and the active enzyme.
Subtilisin E is an alkaline serine protease produced by Bacillus subtilis 168 (Ikemura et al., 1987). The
primary gene product for the enzyme consists of pre-pro-subtilisin,
having a unique 77-residue pro-sequence between the signal peptide
(pre-sequence) and the 275-residue mature sequence. The pro-sequence
has been shown to be essential to produce active subtilisin in vivo and is autoprocessed upon the completion of folding of
prosubtilisin (Ikemura et al., 1988; Ohta et al.,
1990, 1991; Zhu et al., 1989). The autoprocessing of the
propeptide is blocked when any one of the three residues at
the active center (Asp
, His
, and
Ser
) is substituted with other amino acid residues;
Asp
Asn (Zhu et al., 1989), His
Ala (Shinde and Inouye, 1995), and Ser
Ala (Li and Inouye, 1994). Interestingly,
prothiolsubtilisin, in which Ser
was replaced by Cys,
could still autoprocess the propeptide despite the fact that
thiolsubtilisin lost its protease activity against a synthetic
substrate (Li and Inouye, 1994).
The requirement for the propeptide
for the formation of active subtilisin has been demonstrated in
vitro by the finding that the addition of propeptide is essential
to renature subtilisin E denatured by 6 M guanidine HCl (Zhu et al., 1992; Ohta et al., 1991). A number of
mutations within the propeptide have been isolated, which are defective
in producing active subtilisin, indicating that specific amino acid
residues and/or regions in the pro-sequence play important roles in the
process of folding the mature subtilisin (Kobayashi and Inouye, 1992).
Because of its intramolecular requirement for folding into active
subtilisin, the propeptide has been termed an intramolecular chaperone
(Inouye, 1991; Shinde and Inouye, 1993; Shinde et al., 1993).
The existence of such intramolecular chaperones required for the
formation of active enzymes has also been demonstrated for a number of
proteases outside the subtilisin family, such as -lytic protease
from Lysobacter enzymogenes (Silen et al., 1989;
Silen and Agard, 1989) and vacuolar carboxypeptidase Y from Saccharomyces cerevisiae (Winther and Sorensen, 1991; Ramos et al., 1994).
Amino acid substitution mutations in the
pro-sequence that were unable to produce active subtilisin in vivo have been isolated within almost the entire pro-sequence region
(Kobayashi and Inouye, 1992). In the present paper, we selected six of
the 25 mutations previously isolated, and overexpressed them using a T7
expression system in Escherichia coli. These mutant
propeptides were purified to near homogeneity, and their abilities to
refold denatured subtilisin in vitro were examined. We found
that some mutant propeptides were able to refold denatured subtilisin
quite efficiently, while others were very inefficient or incapable of
renaturing unfolded subtilisin. The efficiencies of the mutant
propeptides were found to correlate well to the Kvalues exhibited by these propeptides,
indicating that the binding affinities of the mutant propeptides to
mature subtilisin are directly related to their capacities to function
as intramolecular chaperones.
Six mutant propeptide genes were
cloned into the pET11a vector as follows. Since all those mutations
within the propeptide region were created on the pHI215T vector
(Kobayashi and Inouye, 1992), an NdeI site was first created
at the initiation codon of the gene by PCR using pHI215T containing
desired mutations as a template. The two primers used for PCR were
5`-GCTCTAGACATATGGCCGGAAAAAGCAGTAC-3` and 5`-GGTCGGATCCTTAATATTCATG-3`.
The former, which was used as the 5`-end primer, contained an NdeI site at its 5`-end (underlined). The latter was the
3`-end primer, which annealed to the junction region between the
propeptide and the mature enzyme. Thus, the resulting PCR products
contained the entire propeptide region. The PCR reaction was carried
out with 20 ng of plasmid DNA, each primer at 400 nM, each
dNTP at 0.2 mM, 50 mM KCl, 10 mM Tris-HCl
(pH 8.3), 1.5 mM MgCl, and 0.5 unit of Taq polymerase. The final volume was 50 µl. Denaturation was
carried out at 93 °C for 1 min, annealing at 50 °C for 2 min,
and elongation at 72 °C for 1.5 min. This cycle was repeated 25
times (Saiki et al., 1988). Subsequently, the PCR product
(about 260 base pairs in length) thus obtained was digested with the NdeI restriction enzyme. Since there is another NdeI
site located approximately 30 base pairs upstream of the COOH terminus
of the propeptide, a 200-base pair NdeI-NdeI fragment
that contained most of the propeptide coding region from the NH
terminus resulted from the digestion. The fragments were then
cloned into pET11a-propeptide expression vector, from which the
wild-type NdeI-NdeI propeptide region had been
removed. All constructs were verified by DNA sequencing.
The purification of the propeptides was carried out as follows. The supernatant obtained above was first applied to a cation-ion exchange CM-Sepharose Fast Flow FPLC column, which was equilibrated with 50 mM sodium phosphate buffer (pH 5.0). The propeptide was eluted with a NaCl gradient (0-0.4 M). The propeptide peak was detected, and the pooled fractions were then dialyzed against an excess volume of 50 mM Tris-HCl (pH 8.5). The dialysate was applied to an anion-ion exchange Mono Q-Sepharose Fast Flow column. The protein peak eluted with a 0-0.4 M NaCl gradient was collected.
Figure 1:
Full-length amino acid sequence of the
propeptide of subtilisin E from the NH-terminal -77
position (left) to the COOH-terminal -1 position (right). The large arrow on the COOH terminus
indicates the cleavage site between the propeptide and the mature
sequence. From the COOH terminus, every 10th amino acid residue is
marked by a dot above it. The six single amino acid
substitution mutations used in this study are shown below the
corresponding residues. The open triangle indicates the
position of nonsense mutation, which gives the truncated N59-mer from
the NH
-terminal end of the propeptide. All 20 amino acid
residues are represented by standard single-letter
symbols.
Figure 2:
In vivo subtilisin activity assay. Halo
formation around the colonies was carried out on a casein agar plate as
described under ``Experimental Procedures.'' The plate was
incubated at 37 °C for 1 day and then at room temperature for 6
more days. 215 is a positive control with cells carrying
plasmid pHI215, and 216 is a negative control with cells
carrying plasmid pHI216. A-30T, P-15L, I-48T, K-36E, I-67V, and G-44D represent six amino acid substitution mutations in
the propeptide region (Ala
Thr,
Pro
Leu, Ile
Thr,
Lys
Glu, Ile
Val,
and Gly
Asp,
respectively).
Figure 3: Assays of the protease activity of denatured subtilisin BPN` renatured with the addition of various propeptides. A, the denatured subtilisin BPN` was renatured with the wild-type propeptide as well as with the mutant propeptides as described under ``Experimental Procedures.'' Next an aliquot was taken to assay subtilisin activity with a synthetic substrate, succinyl-Ala-Ala-Pro-Phe-p-nitroanilide. The x axis indicates the incubation time in seconds and the y axis the absorbance at 410 nm. B, relative recovery of the subtilisin activity. From the data in panel A, the initial rate of the reaction was calculated and was compared to that obtained with the wild-type propeptide. Mutant abbreviations are defined in legend to Fig. 2; WT, wild type.
Fig. 4shows the time courses of p-nitroaniline release from the substrate by subtilisin BPN` (Fig. 4A) and subtilisin Carlsberg (Fig. 4B) in the presence of variable concentrations of the wild-type propeptide from subtilisin E. The hyperbolic progress curves for release of p-nitroaniline by active subtilisin in the presence of propeptide of subtilisin E indicate that the propeptide is a slow and tight binding inhibitor for subtilisin BPN` and Carlsberg.
Figure 4:
Progress curves for the onset of slow
binding inhibition of subtilisin BPN` (panel A), subtilisin
Carlsberg (panel B), and subtilisin E (panel C) by
the propeptide of subtilisin E. At the indicated concentrations, the
substrate was premixed with the propeptide before the enzyme was added. A, the final concentrations in the reaction mixture were 50
mM Tris-HCl (pH 7.0), KCl = 0.1 M, CaCl = 1.0 mM, [s-AAPF-pNA] = 0.3
mM, and the reaction was carried out at room temperature with
the subtilisin BPN` at concentration of 2.0
10
mg/ml. B, the buffer condition was the same as above and
the concentration of subtilisin Carlsberg was 3.0
10
mg/ml. C, the final concentrations in
the reaction mixture were 50 mM Tris-HCl (pH 8.0), KCl
= 0.1 M, CaCl
= 1.0 mM,
[s-AAPF-pNA] = 0.5 mM, at 25 °C and the
concentration of subtilisin E was 0.002 mg/ml. D, inhibition
of subtilisin BPN` by the Ile
Thr mutant
propeptide. The mutant propeptide behaves as a rapid equilibrium
competitive inhibitor of subtilisin BPN`. Assay conditions were the
same as in panel A, and the concentrations of the mutant
propeptide are indicated in the figure.
The hyperbolic progress curves for release of p-nitroaniline from s-AAPF-pNA by active subtilisin in the presence of propeptide E indicated that propeptide E is a slow and tight binding inhibitor (Morrison and Walsh, 1988) of subtilisin BPN` and Carlsberg.
Competitive slow binding inhibition generally fits one of the two mechanisms described below (Cha, 1975; Morrison and Walsh, 1988).
Both mechanisms can be described by the same equation (Cha,
1975), where ,
, and
are the
enzymatic hydrolysis rate (at time t), initial rate, and
steady state rate, respectively.
The apparent first order rate constant k, however, has different significance, for a single-step process, as shown by ,
and for a two-step process, as shown in .
The obtained from the on-set progress curves (Fig. 4) is independent of inhibitor concentration [I]
for both subtilisin BPN` and Carlsberg (data not shown). That confirms
that inhibition of subtilisins by pro-peptide E follows ,
similar to inhibition of cathepsin B by its propeptide (Fox et
al., 1992), in which
In contrast, is a function of [I] if
inhibition occurs in a two-step
process.
At constant substrate concentration [S], integration
of gives the following equation, where A and A are the absorbance of the hydrolysis product at
time t and zero.
Hence, and k could be obtained by
non-linear least square fitting of the onset (starting with enzyme)
progress curves (A versus t). The progress curves starting
with substrate also confirmed the slow binding mechanism, but they were
not used here due to the depletion of pro-peptide as a result of
proteolytic digestion during preincubation. Then, K
is estimated by non-linear least square fitting of a set of
versus [I] to the competitive
inhibition equation shown below.
The propeptide is also a good substrate of subtilisins (data not
shown). This observation makes the measurement of K much more complex. A meaningful K
could only
be obtained when the concentrations of propeptide and subtilisin were
carefully balanced. In other words, the chosen propeptide concentration
has to be high enough to slow down the hydrolysis and to maintain a
relatively constant inhibitor concentration, and small enough to give a
measurable steady state velocity.
The K value
of the propeptide of subtilisin E was calculated to be 1.4
10
M against subtilisin BPN` and 1.02
10
M against subtilisin Carlsberg.
Streptomyces subtilisin inhibitor, one of the strongest inhibitors
known of subtilisin, was also found to behave as a slow binding
inhibitor (K
is 0.1 nM against subtilisin
BPN` according to our protocol). However, the propeptide of subtilisin
E is a very weak inhibitor for subtilisin E itself (Fig. 4C). This is probably due to the fact that the
propeptide of subtilisin E is a better substrate of subtilisin E than
of subtilisin BPN` and Carlsberg, and therefore it is digested promptly
by active subtilisin E (Hu, 1994). Since no steady state kinetic
behavior could be reached due to the digestion, no attempts were made
to calculate an accurate K
value for subtilisin E.
For this reason, subtilisin BPN` was used for the present study.
In
the same manner, all six single amino acid substitution mutant
propeptides and the truncated N59-mer propeptide were examined for
their inhibitory behavior toward subtilisin BPN`. The calculated K values are listed in Table 1. Three of the
six single mutant propeptides, Ala
Thr,
Lys
Glu, and Pro
Leu, have comparable K
values with the wild-type
propeptide (1.4
10
M): 1.15
10
, 1.61
10
, and 2.10
10
M, respectively. The other two
propeptides, Gly
Asp and Ile
Val, have approximately 7-8-fold higher K
values than the wild-type propeptide: 11.0
10
and 10.0
10
M for the Gly
Asp and the
Ile
Val propeptides, respectively. The
Ile
Thr mutant gave the most dramatic
result, with a K
value (118
10
M) that is 80 times higher than that for
the wild-type propeptide. This mutant propeptide is no longer a slow
binding inhibitor of subtilisin BPN` (Fig. 5D). The
N59-mer showed no inhibition toward subtilisin BPN`, even at 10
µM concentration (data not shown).
Figure 5:
Plot of folding efficiency of the mutant
propeptides versus the reciprocals of its K. Each filled dot represents
the mutant propeptide or wild-type propeptide (marked accordingly).
Mutant abbreviations are defined in Fig. 2legend.
In the present study, we selected 6 mutations out of 25 amino
acid substitution mutations previously isolated (Kobayashi and Inouye,
1992) for further study. These mutations were originally screened
according to their inabilities to form a halo around colonies on an
agar plate containing casein. E. coli cells producing active
subtilisin form a halo on the plate. By incubating the plate for a much
longer time, three mutants were found to be able to develop halos (see Fig. 2). The propeptides containing these mutations classified
as class I mutations were capable of refolding denatured subtilisin at
a level of 50-80% of the efficiency of the wild-type propeptide.
The K values were found to be well correlated with
the refolding abilities of the class I mutant propeptides;
Ala
Thr showed 80% refolding efficiency with K
of 1.15
10
M, Lys
Glu 60% with K
of 1.61
10
M, and Pro
Leu 54% with K
of 2.10
10
M. It is interesting to note that these three mutations
located in the COOH-terminal half of the propeptide resulted in drastic
amino acid substitutions; a change of charge from +1 to -1
(Lys
Glu), a hydrophobic to a hydrophilic
residue in a cluster of hydrophobic residues (Ala
Thr), and proline to leucine at position -15. It
appears that the propeptide is designed to maintain the chaperone
activity, albeit at a lower level, even with drastic amino acid
substitution mutations in the COOH-terminal region. However, the fact
that the deletion of the COOH-terminal 18 residues (N59-mer) resulted
in the complete loss of refolding ability of the propeptide indicates
that the COOH-terminal region is still essential for the chaperone
function. The second class of mutations (class II; Ile
Val and Gly
Asp) formed no halo
around colonies even after longer incubation, although the propeptides
with the class II mutations were able to refold denatured subtilisin at
a level of 10-20% of the efficiency of the wild-type propeptide.
In this class, the K
values are approximately
7-8 times higher than the K
value of the
wild-type propeptide. The class III mutations, Ile
Thr, had a K
value that was 80 times
higher than that of the wild-type propeptide, and was barely able to
refold denatured subtilisin.
Interestingly, a plot of folding
efficiency of the mutant propeptide versus the reciprocals of
its K leads to a linear relationship (Fig. 5). This suggests that the abilities of the mutant
propeptides to bind to the mature subtilisin are directly related to
their abilities to renature denatured subtilisin. The
propeptide's ability to function as a chaperone for subtilisin
folding is therefore related as to how well the propeptide is able to
bind the mature active subtilisin to inhibit its activity. Neither of
the mutant propeptides nor the wild-type propeptide have significant
amounts of secondary structure, as judged by their circular dichroism
spectra (not shown; see also Shinde et al., 1993). However,
when the wild-type propeptide binds to mature active subtilisin, it
acquires a substantial amount of secondary structure (Shinde et
al., 1993). It has been suggested that the propeptide as a single
turnover catalyst (Hu et al., 1994) exerts its catalytic power
on a late step in the refolding process, in which the interaction
between the propeptide and the mature sequence approximates the
interaction between the propeptide and the active enzyme, i.e. in the transition state of the propeptide catalyzed folding, much
of the secondary and tertiary structure found in the native structure
is already formed. Therefore, the propeptides with the mutations
described in the present paper are likely to become defective in
formation of secondary structure. During the subtilisin folding
process, the interaction between the propeptide and denatured
subtilisin is believed to lead to the formation of secondary structures
in both components. Thus, mutations that inhibit the acquisition of
secondary structure in the propeptide may also render it incapable of
refolding the denatured subtilisin.
Our finding that the propeptides are also substrates for the active enzymes has direct relevance to the notion of a ``single turnover catalyst'' (Hu, 1994). By definition, as a catalyst, the propeptide would catalyze both the refolding and unfolding reactions. Proteolysis of the propeptide renders it ineffective as a catalyst (or indeed as an inhibitor) and traps the mature protein in its refolded state.
Slow binding
inhibition behavior had been observed in the interaction of another
serine protease -lytic protease with its propeptide (Baker et
al., 1992) and in the interaction of the cystein protease
cathepsin B with its propeptide (Fox et al., 1992). Therefore,
the kinetic behavior reported in the present manuscript appears to be
quite general for the inhibition of the active proteases by their
propeptides.
It is possible, although it would clearly be a speculation, that the slow binding inhibition reflects that the final conformation of the propeptide that is bound to the active conformation of the mature enzyme is different from the conformation of the propeptide in the initial encounter complex. Such a conformational change of the bound propeptide would in fact be consistent with the observed kinetics.
It has been recently reported that denatured subtilisin could be refolded in the absence of the propeptide providing that subtilisin is attached to a solid matrix (Hayashi et al., 1993). In that experiment, active subtilisin was first immobilized on CNBr-activated agarose gel and then denatured by 6 M guanidine HCl. Under these conditions, certain sites on the surface of the native subtilisin molecule may preferentially interact with the solid surface, and this interaction might restrict the unfolding process of subtilisin. Furthermore, even if subtilisins were completely denatured in this experiment, the protection of denatured subtilisin at certain sites by the matrix may highly restrict random movement of the molecule being folded, and this may be sufficient to avoid undesired interactions between the secondary structures during the renaturation process. Such an unproductive alternative folding pathway might govern the folding of denatured subtilisin in solution in the absence of the propeptide and perhaps lead to aggregation.
Identification of the step at which the propeptide must exert its influence on the folding pathway of mature subtilisin is a major outstanding issue in this folding mechanism. In the absence of any structural information about the propeptide, the mutational study described in this paper provides a guide to some specific interactions between the propeptide and the mature subtilisin, helping us to understand the molecular mechanism of propeptide-mediated protein folding.