(Received for publication, January 22, 1997, and in revised form, April 21, 1997)
From the Laboratory of Molecular Enzymology, Department of Applied Biological Chemistry, Faculty of Agriculture, Tohoku University, 1-1 Tsutsumidori-Amamiyamachi, Aoba-ku, Sendai 981, Japan
The S1 substrate specificity of porcine pepsin has been altered to resemble that of fungal aspartic proteinase with preference for a basic amino acid residue in P1 by site directed mutagenesis. On the basis of primary and tertiary structures of aspartic proteinases, the active site-flap mutants of porcine pepsin were constructed, which involved the replacement of Thr-77 by Asp (T77D), the insertion of Ser between Gly-78 and Ser-79 (G78(S)S79), and the double mutation (T77D/G78(S)S79). The specificities of the mutants were determined using p-nitrophenylalanine-based substrates containing a Phe or Lys residue at the P1 position. The double mutant cleaved the Lys-Phe(4-NO2) bonds, while wild-type enzyme digested other bonds. In addition, the pH dependence of hydrolysis of Lys-containing substrates by the double mutant indicates that the interactions between Asp-77 of the mutant and P1 Lys contribute to the transition state stabilization. The double mutant was also able to activate bovine trypsinogen to trypsin by the selective cleavage of the Lys6-Ile7 bond of trypsinogen. Results of this study suggest that the structure of the active site flap contributes to the S1 substrate specificity for basic amino acid residues in aspartic proteinases.
Aspartic endopeptidases (EC 3.4.23._) comprise a group of
enzymes whose proteolytic activities are dependent on two aspartyl residues, Asp-32 and Asp-215, in pepsin numbering (1). Mammalian and
fungal enzymes have been extensively characterized, and their three-dimensional structure has been determined at high resolution (2-7). The enzymes of this family are bilobal with a deep and extended
cleft which can accommodate at least seven amino acid residues in the
S4-S3 subsites1
(8). The "flap," an antiparallel
-hairpin loop comprising residues 72 to 82 (pepsin numbering), projects across the cleft forming
a channel into which a substrate binds. Although the enzymes are quite
similar in their three-dimensional structures, there are drastic
differences in the catalytic properties, especially in substrate
specificities. There also have been many studies on the tertiary
structures of aspartic proteinases with peptide-derived inhibitors (4,
5, 9-14), and the relationship between their structures and substrate
specificities has been demonstrated (4, 5, 9-13, 15, 16). According to
these reports, although the hydrogen bonding pattern between the main
chain of inhibitor and enzyme is well conserved in all inhibitor-enzyme
complexes, the differences in the size of subsites, of which residues
make van der Waals contacts with the side chain of inhibitor, may
control the substrate specificities in aspartic proteinases. Several
attempts to examine the structural determinants of substrate
specificities of aspartic proteinases by site-directed mutagenesis have
been made recently (17-23). These provided direct evidence that the subtle differences in the structures of substrate binding sites of
aspartic proteinases were sufficient to alter their substrate specificities.
Aspartic proteinases generally show specificity for the cleavage of the
bond between hydrophobic residues occupying the
S1-S1 subsites. However, fungal enzymes also
have preferences for a Lys residue in the P1 position,
which leads to activation of trypsinogen by cleavage of the
Lys6-Ile7 bond (24-27). The S1
subsite is formed by several hydrophobic residues in the neighborhood
of the catalytic Asp-32 and by the residues on the active site flap.
The sequence alignments of mammalian and fungal enzymes reveal that
Asp-77 and Ser-79 on the flap are conserved in all family members able
to activate trypsinogen, but Asp-77 is replaced by Ser or Thr and
Ser-79 is deleted in those unable to do so (Table I).
Asp-77 is shown to be the binding site to P1 Lys in a
substrate by crystallographic study of penicillopepsin (EC 3.4.23.20)
(11) and site-directed mutagenesis studies of aspergillopepsin I (EC
3.4.23.18) (20, 21) and rhizopuspepsin (EC 3.4.23.21) (22).
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Pepsin (EC 3.4.23.1) is a typical aspartic proteinase produced in the gastric mucosa of vertebrates as a zymogen form. This enzyme has been extensively characterized, and its three-dimensional structure has been determined at high resolution (2, 3, 9). Porcine pepsin, in particular, has been studied as a model to analyze the structure-function relationship of the aspartic proteinases.
The present studies investigated whether the active site flap controls the S1 subsite specificity of aspartic proteinases, and for this we generated several mutants of porcine pepsin by site-directed mutagenesis and analyzed their enzymatic properties. The substitution of Thr-77 by Asp and the insertion of Ser between Gly-78 and Ser-79, corresponding to the fungal enzymes, conferred upon porcine pepsin the ability to hydrolyze the substrate containing a Lys residue at the P1 position. These experiments provide positive evidence of the importance of the active site flap of aspartic proteinases in the S1 subsite specificity.
Porcine pepsin, bovine trypsinogen, and bovine hemoglobin were from Sigma. Pro-Thr-Glu-Phe-Phe(4-NO2)-Arg-Leu was obtained from Calbiochem. Pro-Thr-Glu-Lys-Phe(4-NO2)-Arg-Leu and Ac-Ala-Ala-Lys-Phe(4-NO2)-Ala-Ala-NH2 were custom-synthesized by TANA Laboratories (Houston, TX). Aspergillopepsin I was purified by the method described previously (28).
Strains and PlasmidsEscherichia coli BL21 (DE3) (hsdS gal (lcIts857 ind1 Sam7 nin5 lacUV5-T7 gene)) was the host strain for protein expression. Plasmids, pUC119 and pET12a, were purchased from Takara Shuzo.
Synthesis of Porcine Pepsinogen cDNA and Construction of Expression PlasmidTotal RNA from porcine gastric mucosa was
extracted and purified by the modified acid guanidinium
thiocyanate/phenol/chloroform RNA extraction method (29). Porcine
pepsinogen cDNA was synthesized by reverse transcriptase-polymerase
chain reaction (PCR) (30) using the oligonucleotides
5-CGAACATATGCTCGTCAAGGTCCCGCTGGT-3
and
5
-GGAGGAATTCAGGCTCAGGCCACGGGAGCC-3
, based on cDNA sequence of
porcine pepsinogen reported by Lin et al. (31). Sense primer was designed to delete the signal sequence and introduce a translation initiation codon (Met) for direct expression. Synthesized cDNA was
cloned in pUC119 and sequenced to confirm the absence of an undesired
mutation. The NdeI/SalI fragment was inserted
into pET12a, which was designated as pETPP, to express porcine
pepsinogen under the control of a T7 promoter.
Site-directed mutagenesis was
performed by the method of Kunkel et al. (32). The following
mutagenic primers were used: T77D, 5-CACCTATGGCGACGGATCCATGACAGGC-3
;
G78(S)S79, 5
-TCACCTATGGTACCGGTTCCAGCATGACAG-3
; T77D/G78(S)S79,
5
-CACCTATGGCGACGGATCCAGCATGACAG-3
. In all cases, additional silent
mutations were simultaneously introduced to provide a restriction site
that could be used as verification of mutagenesis. All mutations were
confirmed by dideoxy sequencing before subcloning the mutated cDNA
into pET12a.
E. coli BL21(DE3) cells
harboring the desired mutant plasmid were inoculated into 500 ml of LB
medium with 50 µg/ml ampicillin and cultured at 37 °C until the
OD660 was 0.6;
isopropyl-1-thio--D-galactopyranoside was added to a
final concentration of 0.1 mM, and the incubation was
continued for 4 h. The cells were harvested by centrifugation and
resuspended in 50 ml of TE buffer (50 mM Tris-HCl, pH 8.0, containing 2 mM EDTA). After Triton X-100 was added to the
suspension to a final concentration of 1%, the cells were ruptured by
sonication. The cell homogenate was centrifuged at 6,000 × g for 15 min. The resulting pellets, which contained mostly
porcine pepsinogen as inclusion bodies, were washed by being
resuspended in 10 ml of TE buffer followed by centrifugation. The
washed inclusion bodies were dissolved in 40 ml of TE buffer containing
8 M urea and allowed to stand at room temperature for
2 h, solid NaCl was added to the solution to a final concentration
of 0.5 M, and the pH was adjusted to 10.0 with 1 N NaOH. The solution was dialyzed against 1 liter of 20 mM sodium bicarbonate, pH 10.0, containing 4 M
urea, and the urea concentration was lowered stepwise from 4 M to 2, 1, 0.5, and 0 M every 3 h at room
temperature. The dialyzing buffer was then exchanged for 2 liters of 20 mM sodium phosphate, pH 7.0, and dialysis was continued at
4 °C for 8 h. The dialysate was used as a crude porcine
pepsinogen preparation. Five milliliters of 2 M
glycine-HCl, pH 2.0, was added to 40 ml of this pepsinogen preparation
and incubated at 25 °C for 3 h. The solution was dialyzed against 1 liter of 20 mM sodium acetate, pH 5.0, at
4 °C. The dialysate was applied to an anion-exchange RESOURCE Q
column (Pharmacia Biotech Inc.; 0.64 × 3 cm) and eluted with 20 mM sodium acetate, pH 5.0, with a linear gradient of 0 to
1.0 M NaCl. The active fractions were dialyzed against 10 mM sodium acetate, pH 5.5, and used as purified porcine
pepsinogen. Purity of the enzymes was examined by SDS-PAGE (33), and
their amino-terminal amino acid sequences were determined by the method
of Matsudaira (34).
The circular dichroism (CD) spectra were
measured with a Jasco J-700 spectropolarimeter at room temperature in a
1-mm path length cell. The concentrations of enzymes were adjusted to
0.2 mg/ml in 5 mM sodium acetate, pH 5.0. Contents of the
-helix and
-structure of the enzymes were calculated according to
the SSE-338 program given in Ref. 35.
Proteolytic activity was determined by the method of Ichishima (36) in 50 mM sodium citrate, pH 2.0, containing 1.25% acid-denatured hemoglobin at 30 °C. Trypsinogen activation was determined as described previously (20).
Kinetic Characterizations of Mutant Porcine PepsinsThree chromogenic peptide substrates, Pro-Thr-Glu-Phe-Phe(4-NO2)-Arg-Leu (peptide A), Pro-Thr-Glu-Lys-Phe(4-NO2)-Arg-Leu (peptide B), and Ac-Ala-Ala-Lys-Phe(4-NO2)-Ala-Ala-NH2 (peptide C), were used as substrates for kinetic analyses. Assays with peptides A and B were performed as described by Dunn et al. (37) and with peptide C by the method of Hofmann and Hodges (38). The kinetic constants, Km and Vmax, were determined from plots of initial rates versus substrate concentrations with ranges of 0.010-1.000 mM for all substrates. Values for kcat were derived from Vmax = kcat [E], where [E] is the enzyme concentration.
Product AnalysisPurified enzymes (0.35 µg) were incubated in total volume of 100 µl of 20 mM sodium acetate, pH 3.5, containing 10 nmol of each peptide substrate (peptides A, B, and C) for 8 h at 37 °C (enzyme/substrate, 1/1,000 mol/mol). Cleavage products were separated by reverse-phase high performance liquid chromatography on a TSK ODS-120T column (4.0 × 250 mm, Tosoh, Tokyo, Japan) and confirmed by amino acid sequence analysis.
pH Dependence StudiesThe kinetic parameters of the double mutant for three peptide substrates (peptides A, B, and C) were determined at pH 2.55-6.0 in 0.1 M sodium acetate. The enzyme concentrations used in these experiments were 1.0-115 nM, 28.8-288 nM, and 28.8-230 nM for peptides A, B, and C, respectively.
The structure of porcine pepsinogen cDNA was reported earlier by Lin et al. (31). Therefore, porcine pepsinogen cDNA was cloned by reverse transcriptase-PCR, using total RNA from porcine gastric mucosa as a template. PCR primers were designed for direct expression in the E. coli pET system. The amplified cDNA was dideoxy-sequenced to verify whether it was synthesized correctly. The sequence was identical with that reported by Lin et al. (31) except for the substitution of Tyr-242 by Asp. Porcine pepsin has been sequenced in several laboratories by protein and cDNA sequencing (39, 40), and these studies indicated that there are two variants at position 242 of amino acid sequence (Asp or Tyr). The electrospray mass spectrometry of porcine pepsin also demonstrated the existence of two variants (41). Hence, the cDNA obtained in this study seemed to be correct and available for expression studies.
The E. coli cells harboring pETPP produced pepsinogen as an
inclusion body under the control of the T7 promoter. The inclusion body
was purified by washing with buffer and then refolded by solubilization
with 8 M urea and subsequent dialysis under alkaline pH as
described under "Experimental Procedures." Porcine pepsin was
purified from the refolded pepsinogen preparation by acidification and
subsequent chromatography on a RESOURCE Q column and gave a single band
on SDS-PAGE (Fig. 1, lane 2). The
NH2-terminal sequence of recombinant pepsin was found to be
predominantly Ile-Gly-Asp-Glu-Pro-Leu-Glu-Asn- ... which is known
as the NH2-terminal sequence of porcine pepsin (39), and a
minor sequence of Ala-Ala-Leu-Ile-Gly-Asp-Glu-Pro- ... was also
detected; the same results were reported in the activation of native
porcine pepsinogen by Kageyama and Takahashi (42). The secondary
structures of native and recombinant porcine pepsin were analyzed by CD
spectrometry. The CD spectral data showed that the spectrum of the
recombinant enzyme was essentially superimposable on that of the native
enzyme (data not shown). The native and recombinant enzymes were almost
equal in specific activity for hydrolysis of acid-denatured hemoglobin
(82 and 83 millikatals/kg, respectively).
Molecular Properties of Mutant Porcine Pepsins
The mutant porcine pepsins, T77D, G78(S)S79, and T77D/G78(S)S79, were purified by the same method as wild-type pepsin, and the purities of the enzymes were judged by SDS-PAGE (Fig. 1). The NH2-terminal sequences of the mutants were the same as that of wild-type enzyme. The secondary structures of recombinant wild-type and mutant pepsins were analyzed by CD spectrometry to determine whether localized or global changes of structures were induced by the mutations. The CD spectra data showed that the spectra of the mutants were essentially superimposable on that of the wild-type enzyme (data not shown). These results suggest that no major conformational alterations occurred in the mutant enzymes.
Enzymatic Properties of Mutant Porcine Pepsins for Protein SubstratesProteolytic activities for acid-denatured hemoglobin
and trypsinogen-activating activities due to limited proteolysis of the Lys6-Ile7 bond of trypsinogen were measured
with wild-type and mutant pepsins. The specific activities of mutant
enzymes, T77D, G78(S)S79, and T77D/G78(S)S79, for hemoglobin hydrolysis
were determined to be 53, 8, and 50 millikatals/kg, respectively. Each
mutant enzyme effectively hydrolyzed hemoglobin although the activity
of G78(S)S79 mutant was 10 times lower than that of wild-type enzyme.
The specific activities of wild-type, T77D, and T77D/G78(S)S79 enzymes
for trypsinogen activation were 0.04, 0.01, and 18.5 microkatals/ml, respectively, and no detectable activity was found for G78(S)S79 enzyme, which indicates that the activity of trypsinogen activation was
significantly increased only by the double mutant. However, it was
still 300-fold less than the activity of aspergillopepsin I. SDS-PAGE
analysis showed that trypsinogen was converted to trypsin by
T77D/G78(S)S79 mutant alone (Fig. 2) and the cleavage site was found to be Lys6-Ile7 by
NH2-terminal sequencing. These results indicated that the double mutation in the active site flap altered substrate specificity of porcine pepsin to those of fungal aspartic proteinases,
aspergillopepsin, penicillopepsin, and rhizopuspepsin.
Kinetic Properties of Mutant Pepsins for Peptide Substrates
To probe the effects of the mutations on substrate
specificity for the P1 residue, we used the peptide
substrates, Pro-Thr-Glu-Phe-Phe(4-NO2)-Arg-Leu (peptide A),
Pro-Thr-Glu-Lys-Phe(4-NO2)-Arg-Leu (peptide B), and Ac-Ala-Ala-Lys-Phe(4-NO2)-Ala-Ala-NH2 (peptide
C). Peptide A was cleaved at the Phe-Phe(4-NO2) bond by
each enzyme, which indicated that mutant enzymes also prefer
hydrophobic residues in P1 and P1 in the
substrate. The T77D/G78(S)S79 mutant hydrolyzed
Lys-Phe(4-NO2) bonds in peptides B and C, while wild-type
enzyme cleaved mainly at Glu-Lys and Phe(4-NO2)-Ala bonds
in peptides B and C, respectively. The T77D mutant cleaved at the
Lys-Phe(4-NO2) bond in peptide B, but at the
Phe(4-NO2)-Ala bond in peptide C. There was no cleavage in
peptides B and C by G78(S)S79 mutants. These results indicate that the
T77D/G78(S)S79 mutant acquired the ability to recognize a Lys residue
at the P1 position although the enzyme still exhibited a
preference for hydrophobic residues at the P1 position.
From kinetic determination of the
kcat/Km (catalytic
efficiency) one can obtain the second-order rate constant for
conversion of a substrate to a product (43). Combining effects due to
substrate binding and transition state stabilization, this parameter is useful for assessing altered substrate specificity. Differences in
log(kcat/Km) provide an
accurate measure of the lowering of the transition state activation
energy (
GT
). The kinetic
parameters for hydrolysis of Phe-Phe(4-NO2) and Lys-Phe(4-NO2) bonds were determined from the initial rate
measurements, and the results are listed in Table II.
The mutations led to a 4- to 130-fold decrease of
kcat/Km compared to wild-type enzyme for peptide A. However, the
kcat/Km value for the double
mutant was larger than those of single mutants. Although the
kcat/Km values of the double
mutant for peptides B and C were 50 and 30 times smaller than that for
peptide A, respectively, the double mutation led to hydrolysis of the
Lys-Phe(4-NO2) bonds in the former two peptides. The double
mutant showed a similar ratio of
kcat/Km for peptide C over
peptide A to aspergillopepsin I, although the preference for peptide B
was significantly different between the double mutated pepsin and
aspergillopepsin I. These results indicated that fungal aspartic
proteinase-like specificity can be introduced into porcine pepsin.
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To
obtain information on recognition of a Lys residue at the
P1 position by the T77D/G78(S)S79 double mutant, the
kinetic parameters for this double mutant were determined at various pH values using three peptide substrates containing a Phe or Lys in
P1 (Fig. 3). In all substrates, the
Km values were pH independent, and above pH 4 the
values of kcat and
kcat/Km were controlled by
the dissociation of a carboxyl group with a pKa of
about 5. Below pH 4, however, the kcat and
kcat/Km values for the
P1 Lys substrates (peptides B and C) decreased as pH
declined, while there was no significant change in
kcat and
kcat/Km values for the
P1 Phe substrate (peptide A).
The pH-dependent changes of substrate preference are shown
in Fig. 4. The P1 Lys/Phe preference
increased as pH rose, which indicates that the dissociation of a
carboxyl group as Asp-77 may affect the preference of a Lys residue at
the P1 position.
The double mutation in porcine pepsin, the substitution of Thr-77
by Asp, and the insertion of Ser between Gly-78 and Ser-79 (T77D/G78(S)S79), successfully altered the S1 subsite
specificity of pepsin to those of fungal aspartic proteinases. Some
fungal aspartic proteinases, aspergillopepsin I, penicillopepsin,
rhizopuspepsin, and others, are able to activate trypsinogen by
cleavage of its Lys6-Ile7 bond, showing an
affinity for the anionic lysine side chain in S1 (26). It
is known that Asp-77 (pepsin numbering) of these enzymes is the binding
site to the Lys residue in P1 by means of site-directed
mutagenesis of aspergillopepsin I (20, 21) and rhizopuspepsin (22), as
pointed out for the case of inhibitor binding to penicillopepsin (11).
In porcine pepsin, the hydrophobic side chain can be accommodated in
the S1 subsite, while basic residue cannot. As expected,
the double mutant pepsin was able to accept the lysine residue in
P1, which led to trypsinogen activation, although it still
showed a preference for the hydrophobic residue in P1.
However, the single mutants, T77D and G78(S)S79, were not able to
hydrolyze the substrates containing a Lys residue in P1 and
also exhibited lower catalytic efficiency
(kcat/Km) for the
P1 Phe substrate than those of wild-type and double mutant enzymes. The double mutant exhibited a similar preference for peptide C
as did aspergillopepsin I, while for peptide B the catalytic efficiency
of double mutated pepsin was about 20-fold lower than that of
aspergillopepsin I; this was mainly due to the smaller Km values of aspergillopepsin I for peptides A and B than those of the double mutated pepsin. The differences in the substrate affinity may be a consequence of alterations in the structures of other substrate binding sites (S4,
S3, S2, S2, and S3
)
than the S1 and S1
subsites.
Fig. 5 shows the structures of S1 subsites
of human pepsin and penicillopepsin complexed with pepstatin and
statine-based inhibitor, Iva-Val-Val-LySta-OEt (Iva, isovaleryl; LySta,
4S,3S-4,8-diamino-3-hydroxyloctanoic acid),
respectively. The hydroxy group of the transition state analogue,
statine
(4S,3S-4-amino-3-hydroxyl-6-methylheptanoic acid)
or LySta, is hydrogen-bonding to the oxygen atoms of two catalytic
aspartyl side chains, and the positions of the analogue and catalytic
apparatus are essentially superimposable in both complexes despite the
differences in the flap structures. The flap structure of human pepsin
is included in the typical "class 2" -hairpin loop, while that
of penicillopepsin is an anomalous structure, which has been included
in the "class 3"
-hairpin loop according to Milner and Poet
(44). Schematic models of the flap structure of human pepsin and
penicillopepsin show that Ser-79 of penicillopepsin is inserted while
human pepsin is not (Fig. 6). However, the hydrogen
bonds between the main chain of the inhibitor and the enzyme are well
conserved in both complexes, which may contribute to the proper
orientation of the scissile peptide bond. The 77th amino acid, Thr in
pepsin and Asp in penicillopepsin, forms hydrogen bonds to the
P2 NH and the carbonyl group of main chain in the
inhibitor. In the penicillopepsin complex, the side chains of Asp-77
and Ser-79 form additional hydrogen bonds to the side chain of the Lys
residue at P1.
In this study, the insertion of Ser between Gly-78 and Ser-79 of
porcine pepsin may have caused an alteration in the direction of the
Thr-77 side chain and have disabled the side chain of Thr-77 from
hydrogen bonding to the main chain of the substrate. These would result
in overall decreases in catalytic efficiencies for all substrates in
the G78(S)S79 mutant. In the T77D mutant, the presence of the longer
Asp-77 side chain may allow the flap to bind more loosely to the
substrate and result in a decrease in the number of van der Waals
contacts between enzyme and substrate; this would lead to a reduction
in catalytic efficiency. However, the T77D mutant enzyme hydrolyzed the
Lys-Phe(4-NO2) bond in
Pro-Thr-Glu-Lys-Phe(4-NO2)-Arg-Leu (peptide B) although its
catalytic efficiency was very low, whereas the Glu-Lys bond was the
main cleavage site in wild-type pepsin. This would be due to the
electrostatic repulsion between Glu of the substrate and Asp-77 of the
T77D mutant enzyme, and it would not be the positive recognition of
anionic Lys residue in P1. The wild-type and T77D mutant
enzymes cleaved the Phe(4-NO2)-Ala bond in
Ac-Ala-Ala-Lys-Phe(4-NO2)-Ala-Ala-NH2 (peptide
C) due to the strong interaction between the Phe(4-NO2)
residue of the substrate and S1 hydrophobic pocket of the
mutants. The double mutated pepsin exhibited higher catalytic
efficiency (kcat/Km) for the
P1 Phe substrate than did the single mutants. The double mutant would have a flap structure and hydrogen bond network between Asp-77 and the main chain of a substrate similar to those of the fungal
aspartic proteinases. In the double mutant, the scissile peptide bond
of the substrate may lie at a more stable position. Furthermore, the
double mutant effectively hydrolyzed the Lys-Phe(4-NO2) bond in peptides B and C, due to the additional two hydrogen bonds; the
side chains of Asp-77 and inserted Ser in the mutant may be hydrogen-bonded to the -amino group of the Lys residue in
P1 as fungal enzymes. In a previous study on
aspergillopepsin I (21), the effects of the substitution of Ser-79 by
Ala (S79A) and the deletion of Ser-79 (
S79; corresponding to porcine
pepsin) on the substrate specificity were analyzed. The P1
Lys/Phe preference of the
S79 mutant was reduced more significantly
than the S79A enzyme. Therefore, in mutant pepsins the insertion of Ser
is important for the orientation of the side chain at position 77, although this Ser may also contribute to the hydrogen acceptor in the
hydrogen-bonding interaction.
The effects of the dissociation of the -carboxyl group of Asp-77 in
double mutated pepsin on the recognition of Lys residue at the
P1 position were examined by pH-dependent
hydrolysis of two types of peptide substrates containing Phe or Lys at
P1. The major differences in pH activity profiles were the
kcat/Km values below pH 4. These values for P1 Lys substrates (peptides B and C)
decreased as the pH dropped, while there was no significant change for
P1 Phe substrate (peptide A). The Km
values were pH independent in all cases. These results are different from those of the studies that the existence of Lys or Arg residues at
the P4, P3, P2, P3
,
P4
, and P5
positions in the substrates influences the Km values with little effect on
kcat in porcine pepsin (45, 46); the ionic
interactions between the basic residues in the substrate and the
side-chain carboxylates in the S4, S3,
S2, S3
, S4
, and S5
subsites contribute to substrate affinity. In double mutated pepsin, it
is likely that the dissociation of Asp-77 side chain contributes to the
stabilization of the transition state when substrates contain Lys
residue at the P1 position rather than substrate affinity.
The electrostatic interaction between Asp-77 side chain and
P1 Lys of the substrates could play an important role in
the recognition of Lys residue at the P1 position. In aspergillopepsin I, however, the substitution of Asp-77 by Asn hardly
affected the P1 Lys/Phe preference compared with wild-type enzyme, whereas the substitution of Asp-77 by other amino acids (Glu,
Ser, and Thr) and the deletion of Ser-79 reduced the P1 Lys/Phe preference with the decrease in the kcat
values for the P1 Lys substrates. This indicates that the
recognition of basic P1 side chain is not dependent on the
specific electrostatic interactions between P1 side chain
and Asp-77 side chain of the enzyme (21). From a site-directed
mutagenesis study of rhizopuspepsin, Lowther et al. (22)
concluded that the presence of Asp at position 77 has the potential to
establish an extensive hydrogen-bonding network between the enzyme and
the substrate containing Lys residue at the P1 position. In
the double mutated pepsin, although the dissociation of the
-carboxyl group of Asp-77 would be dispensable to accommodate the
Lys residue in the S1 pocket, it enhances the stabilization of the transition state complex when substrates contain a Lys residue
at the P1 position. This may be due to the formation of the
electrostatic interaction or the changes of the hydrogen-bonding pattern between Lys residue in P1 and Asp-77 of the mutant
pepsin as the pH is higher.