Department of Food Science, University of Guelph, Guelph,Ontario N1G 2W1, Canada
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Abstract |
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Keywords: enzyme stability/N-terminal fragment/pepsin/pepsinogen/pH
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Introduction |
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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 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 ß-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 N-terminal 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.
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Materials and methods |
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The expression plasmid pTFP1000, which contains the gene for wild-type porcine pepsinogen fused to the 3' end of the 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 uracil-containing 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';
240246/+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);
240246/+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 (1x M9 salts, 0.2% casamino acids, 0.5% glucose, 1 mM magnesium 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 TrisHCl buffer pH 7.5, for 2 h at 4°C, filter sterilized and stored at 4°C. Purity was assessed by SDSPAGE, 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 0.010.2 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 Trismalate 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.
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Results and discussion |
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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 0.522.88 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 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.
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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
.
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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 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 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 N- or 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. 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 N-terminal portion is relatively easily displaced from its original position. If this N-terminal portion moves away from its original position, a ß-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 ß-sheet 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).
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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.
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), N-frag 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
-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.
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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
).
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Conclusion |
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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.
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Notes |
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Acknowledgments |
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References |
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Received February 26, 2001; revised May 9, 2001; accepted June 15, 2001.