©1995 by The American Society for Biochemistry and Molecular Biology, Inc.
Functional Analysis of the Propeptide of Subtilisin E as an Intramolecular Chaperone for Protein Folding
REFOLDING AND INHIBITORY ABILITIES OF PROPEPTIDE MUTANTS (*)

(Received for publication, July 6, 1995; and in revised form, August 6, 1995)

Yuyun Li (1) Zhixiang Hu (2) Frank Jordan (2) Masayori Inouye (1)

From the  (1)Department of Biochemistry, Robert Wood Johnson Medical School, University of Medicine and Dentistry-New Jersey, Piscataway, New Jersey 08854 and the (2)Department of Chemistry, Rutgers, State of University of New Jersey, Newark, New Jersey 07102

ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES

ABSTRACT

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(i) values against subtilisin BPN` were 1.6 times 10M, 1.2 times 10M, and 2.1 times 10M, respectively, almost identical to the K(i) value exhibited by the wild-type propeptide (1.4 times 10M). In contrast, Ile Val and Gly Asp were able to refold denatured subtilisin BPN` with only 18 and 13% efficiencies and had K(i) values of 10 and 11 times 10M, respectively. The Ile Thr mutant propeptide was unable to refold denatured subtilisin BPN` and gave a 100-fold higher K(i) (118 times 10M) 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.


INTRODUCTION

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^1 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 alpha-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 K(i)values 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.


EXPERIMENTAL PROCEDURES

Materials

All restriction enzymes and T4 DNA ligase were purchased from New England Biolabs Inc. or Life Technologies Inc. A Sequenase kit was purchased from U. S. Biochemical Corp., and thermostable Taq DNA polymerase for PCR^2 was obtained from Perkin-Elmer. All enzymes were used as recommended by the manufacturers. All oligonucleotides were synthesized on a 0.2-µmol scale on an Applied Biosystems model 380B synthesizer using Applied Biosystems reagents. Purification of oligonucleotides was carried out on OPC cartridges supplied by Applied Biosystems. IPTG was from Gold Biotechnology, Inc. Guanidine HCl, CM-Sepharose Fast Flow cation-ion exchanger and Mono Q-Sepharose Fast Flow anion-ion exchanger, succinyl-Ala-Ala-Pro-Phe-p-nitroanilide (subtilisin substrate) and subtilisin BPN` (protease type XXVII, Nagarse) were purchased from Sigma. Casein was from Difco. Type VM membrane filters (0.05-µm pore size) were from Millipore, Inc.

Strains and Media

E. coli strain BL21(DE3) was a bacteriophage DE3 lysogen in which the phage DE3 was stably integrated into the chromosomal DNA and used as a host cell strain in a T7 expression system (Studier et al., 1990). Strain JA221 (Nakamura et al., 1982) was used for in vivo protease assay, i.e. the halo-formation assay. Strain Cl83 is a recA derivative of JM83 and was used in the subcloning steps. Cultures were carried out in M9 medium (Maniatis et al., 1982) supplemented with 2% casamino acids (Difco), 0.4% glucose, 0.02% MgSO(4), 0.05 mg/ml tryptophan, 0.5 µg/ml vitamin B(1), and 50-200 µg/ml ampicillin.

In Vivo Subtilisin Protease Assay

Subtilisin expression vectors (pINIIIA3) (Ikemura et al., 1987) containing the wild-type propeptide or mutant propeptides were transformed into strain JA221. Overnight cultures (1 ml) from single colonies were diluted 1000 times, and 1 µl of each diluted cell culture was spotted on agar plates containing 2% casein (Takagi et al., 1989). After the plates were incubated at 37 °C overnight, they were incubated at room temperature for 6 more days. Only those cells producing active subtilisin can form a clear halo around the spotted colony by hydrolyzing casein added in the agar plate.

Construction of T7 Expression System

The genes for mutant prosubtilisins (Ser Cys or Ser Ala) have been cloned into T7 expression vector pET11a (Li and Inouye, 1994). To express the propeptide from the same vector, a stop codon was introduced immediately after the propeptide sequence by site-directed mutagenesis, using oligonucleotide 5`-CATGAATATTAGCAATCTG-3` (the stop codon is underlined). The wild-type propeptide expressed from this vector contained an extra methionine at the NH(2)-terminal end.

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(2), 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(2) 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.

Protein Expression and Purification

The cloned gene expression was induced as described before (Li and Inouye, 1994). Both wild-type and mutant propeptides were expressed in the soluble fraction as well as in the inclusion body fraction. The inclusion bodies from a 1-liter culture were solubilized in 15 ml of 6 M guanidine HCl. After overnight incubation at 4 °C, insoluble materials were removed by centrifugation at 90,000 times g for 40 min. The supernatant was then dialyzed against an excess volume of 50 mM sodium potassium phosphate buffer (pH 5.0) at 4 °C. The dialysate was centrifuged at 90,000 times g for 40 min to remove precipitates, and the propeptide was recovered in the supernatant.

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.

In Vitro Renaturation of Denatured Subtilisin by Propeptides

Both subtilisin and the propeptides were dissolved in 10 mM sodium phosphate buffer (pH 5.8) containing 6 M guanidine HCl, and the solutions were incubated at room temperature or 4 °C for several hours. The two solutions were mixed in different ratios, keeping the final concentration of subtilisin at 15 µM. Mixtures (100 µl) were then spotted onto a type VM membrane disk and subjected to dialysis against 500 ml of 10 mM sodium phosphate buffer (pH 7.0) containing 0.5 M (NH(4))(2)SO(4), 1 mM CaCl(2) at 4 °C for 3-4 h. The samples were recovered from the membrane disk. Subtilisin activity was measured using 0.13 mM synthetic peptide substrate succinyl-Ala-Ala-Pro-Phe-p-nitroanilide in 50 mM Tris-HCl (pH 8.5), 1 mM CaCl(2) in a final volume of 0.25 ml. The renatured subtilisin samples were first treated with L-1-tosylamido-2-phenylethyl chloromethyl ketone-treated trypsin (1/100 of subtilisin by weight) for 30 min at room temperature to digest the propeptide, which otherwise would inhibit the active mature subtilisin. The reactions were carried out on a 96-well microplate culture dish and initiated by the addition of the substrate. The dish was maintained at room temperature for 20 min. The absorbance of p-nitroaniline produced during the incubation was measured at 410 nm by an automatic microplate reader (model 3550-UV, Bio-Rad).

Determination of the Inhibition Constants

A COBAS-Bio centrifugal UV-visible analyzer (Roche Diagnostics) was used for kinetic data collection, and the slope of the linear region of the absorbance versus time curve was used as the initial velocity. The steady state kinetic constants K(m), V(max), and K(i) were always determined by simultaneously using the same enzyme solutions at the same time on the COBAS-Bio at room temperature. The release of p-nitroaniline was monitored at 400 nm to minimize the absorption due to the substrate.


RESULTS

In Vivo Subtilisin Activity Assay

Previously, using localized PCR random mutagenesis, a total of 25 single amino acid substitution mutations were isolated that affected the production of active subtilisin in vivo (Kobayashi and Inouye, 1992). The cells harboring plasmids containing these mutant prosubtilisin genes formed no or very thin halos around colonies on casein plates. In this study, we selected the following six mutants from the 25 propeptide mutants for further studies: Ile Val, Ile Thr, Gly Asp, Lys Glu, Ala Thr, and Pro Leu (see Fig. 1). The reasons for selecting these six mutants from the 25 mutants previously isolated are as follows. (a) There are three short hydrophobic regions H1, H2, and H3 in the propeptide, which are considered to play important roles in the folding of prosubtilisin (Kobayashi and Inouye, 1992). The H1 and H2 regions were hot spots for mutants: 4 mutants from H1 and 7 from H2. One mutant Ile Val from H1 and another Ala Thr from H2 were selected for further analysis in this report. (b) Two mutants, Gly Asp and Lys Glu, were selected because of the charge changes. (c) At Ile, there were three mutations, one of which (Ile Thr) was selected. (d) There is only one proline residue in the propeptide. Therefore, Pro Leu was also chosen for further study. (e) Four mutations at the signal peptide cleavage site were not included in this study because they are more likely to inhibit secretion of prosubtilisin rather than its folding. We first reexamined their abilities to form halos on a casein agar plate. None of the cells carrying propeptide mutations formed any halos after a single overnight incubation of the plate at 37 °C (not shown). However, after an additional 6 days of incubation at room temperature, some of them developed halos of different sizes as shown in Fig. 2. Note that cells expressing an active site mutant (Ala Asn) subtilisin, pHI216, did not form halo even after the 6-day incubation. Similarly, cells with the Ile Val, Ile Thr, and Gly Asp mutations did not develop halos. However, cells with the Lys Glu, Ala Thr, and Pro Leu mutations did develop clearly discernible halos. The halo sizes are considered to be related to the ability of cells to produce active subtilisin, which requires secretion of prosubtilisin across the membrane and its subsequent folding outside the cytoplasmic membrane. On the basis of these considerations, the mutant propeptides can be ranked for their abilities to form the active subtilisin as follows: Lys Glu > Ala Thr > Pro Leu > Ile Val, Ile Thr, Gly Asp.


Figure 1: Full-length amino acid sequence of the propeptide of subtilisin E from the NH(2)-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(2)-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).



Expression and Purification of Mutant Propeptides

E. coli strain BL(DE3) carrying the pET11a with a mutant propeptide gene was able to produce the propeptide at a level of more than 50% of total cellular proteins in the presence of 1 mM IPTG (not shown). The propeptide thus produced was equally distributed between the cytoplasmic fraction and the inclusion bodies (not shown). The propeptide was purified from inclusion bodies as described in detail under ``Experimental Procedures.'' After passage through two ion exchange columns, the wild-type propeptide was purified to higher than 99% homogeneity as judged by 17% SDS-polyacrylamide gel electrophoresis (not shown). The other six mutant propeptides were purified in the same manner to a similar homogeneity as the wild-type propeptide. During the gene manipulation, we also constructed a clone that expressed a truncated propeptide consisting of only 59 amino acid residues extending from Leu to the amino-terminal Met. This truncated propeptide was designated as N59-mer and was also purified to homogeneity.

In Vitro Renaturation of Denatured Subtilisin by Mutant Propeptides

Previously, we synthesized the wild-type propeptide of subtilisin E and BPN` and demonstrated that they were able to renature denatured subtilisin E as well as subtilisin BPN` and Carlsberg in vitro, although the efficiency of renaturation was low (Ohta et al., 1991; Zhu et al., 1992). We are now able to produce the propeptide in vivo in large quantities. We reexamined the ability of the wild-type propeptide of subtilisin E to renature denatured subtilisin by the drop-dialysis method (see ``Experimental Procedures''). Since subtilisin E and subtilisin BPN` are highly homologous (88.5% identity in the proregion and 86.2% in the mature region), in this study we examined the renaturation activity of the wild-type propeptide of subtilisin E toward denatured subtilisin BPN`. The reason for using subtilisin BPN` is that it digested the propeptides more slowly than does subtilisin E (Hu, 1994). Subtilisin BPN` was directly dissolved in 6 M guanidine HCl at pH 5.8 to avoid autolysis of subtilisin BPN` during the treatment. Renaturation was carried out with a 2:1 molar ratio of propeptide:subtilisin BPN`, as described under ``Experimental Procedures.'' As shown in Fig. 3A, the wild-type propeptide was able to lead to efficient recovery of subtilisin activity. No activity was recovered in the absence of the propeptide (data not shown). From the activity shown in Fig. 3A, the efficiency of the activity recovery was estimated to be 25% of the activity prior to denaturation of subtilisin BPN`. Fig. 3A also shows the abilities of the six other mutant propeptides for refolding of denatured subtilisin BPN`. From the initial rates measured from the figure, the refolding efficiencies of the mutant propeptides are replotted in Fig. 3B, which are in general agreement with the order of the ability to form a halo by the mutant propeptides shown in Fig. 2. However, it is rather surprising that the Ala Thr propeptide has as high as 80% efficiency compared to that of the wild-type propeptide in the refolding activity. On the casein agar plate, the effect of the Ala Thr mutation might have affected not only the refolding process but also the secretion of the mutant prosubtilisin. Similarly, the Ile Val and Gly Asp propeptides were able to refold the denatured subtilisin BPN` at significant levels (18 and 13% of the wild-type level, respectively; Fig. 3, A and B). This is also unexpected from the halo formation shown in Fig. 2. Among the mutant propeptides, only the Ile Thr propeptide (Fig. 3) and the truncated N59-mer propeptide were unable to refold the denatured subtilisin BPN`.


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.



Binding of Mutant Propeptides to Subtilisins

The above results indicate that the amino acid substitution mutations in the propeptide isolated previously indeed are defective in their abilities to refold the denatured subtilisin. Since these mutations are considered to cause structural changes in the propeptide, we next examined their abilities as inhibitors of subtilisin activity. It has been shown previously that the wild-type propeptides chemically synthesized function as competitive inhibitors (Ohta et al., 1991; Zhu et al., 1992).

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(2) = 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 times 10 mg/ml. B, the buffer condition was the same as above and the concentration of subtilisin Carlsberg was 3.0 times 10 mg/ml. C, the final concentrations in the reaction mixture were 50 mM Tris-HCl (pH 8.0), KCl = 0.1 M, CaCl(2) = 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 , (0), and (s) 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 (0) 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, (0) 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(0) are the absorbance of the hydrolysis product at time t and zero.

Hence, (s) 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(i) is estimated by non-linear least square fitting of a set of (s)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(i) much more complex. A meaningful K(i) 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(i) value of the propeptide of subtilisin E was calculated to be 1.4 times 10M against subtilisin BPN` and 1.02 times 10M 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(i) 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(i) 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(i) values are listed in Table 1. Three of the six single mutant propeptides, Ala Thr, Lys Glu, and Pro Leu, have comparable K(i) values with the wild-type propeptide (1.4 times 10M): 1.15 times 10, 1.61 times 10, and 2.10 times 10M, respectively. The other two propeptides, Gly Asp and Ile Val, have approximately 7-8-fold higher K(i) values than the wild-type propeptide: 11.0 times 10 and 10.0 times 10M for the Gly Asp and the Ile Val propeptides, respectively. The Ile Thr mutant gave the most dramatic result, with a K(i) value (118 times 10M) 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(i). Each filled dot represents the mutant propeptide or wild-type propeptide (marked accordingly). Mutant abbreviations are defined in Fig. 2legend.




DISCUSSION

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(i) 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(i) of 1.15 times 10M, Lys Glu 60% with K(i) of 1.61 times 10M, and Pro Leu 54% with K(i) of 2.10 times 10M. 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(i) values are approximately 7-8 times higher than the K(i) value of the wild-type propeptide. The class III mutations, Ile Thr, had a K(i) 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(i) 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 alpha-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.


FOOTNOTES

*
Work at Rutgers was supported by the Charles and Johanna Busch Biomedical Grant. This work was also supported in part by a grant from Ajinomoto Co., Ltd. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore by hereby marked ``advertisement'' in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

(^1)
The numbering of the propeptide starts from Tyr and extends to Met, i.e. from the carboxyl terminus toward the amino terminus. The mutant N59-mer extends from Leu to Met.

(^2)
The abbreviations used are: PCR, polymerase chain reaction; IPTG, isopropyl-beta-D-thiolgalactopyranoside; s-AAPF-pNA, succinyl-Ala-Ala-Pro-Phe-p-nitroanilide.


ACKNOWLEDGEMENTS

We thank Dr. Ujwal Shinde for critical reading of the manuscript, helpful suggestions, and preparation of Fig. 3A.


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