(Received for publication, April 6, 1995; and in revised form, June 2, 1995)
From the
Pepsin contains a single lysine residue which protrudes from the
enzyme's surface, behind the active site cleft, on the C-terminal
domain. Mutations of pepsin by site-directed mutagenesis of the Lys-319
residue were generated to study the structure-function relationships.
Kinetic parameters, pH activity profiles, along with conformational
analysis using circular dichroism (CD), and molecular modelling were
examined for the wild-type (non-mutant) and mutant enzymes. The pepsin
mutations, Lys-319 Met and Lys-319
Glu, resulted in a
progressive increase in the K
and similar
decrease in k
, respectively, as well as being
denatured at a lower pH than the wild-type pepsin. CD analysis
indicated that mutations at Lys-319 resulted in changes in secondary
structure fractions which were reflected in changes in enzymatic
activity as compared to the wild-type pepsin, i.e. kinetic
data and pH denaturation study. Molecular modelling of mutant enzymes
indicated differences in flexibility in the flap loop region of the
active site, the region around the entrance of the active site cleft,
subsite regions for peptide binding, and in the subdomains of the
C-terminal domain when compared to the wild-type enzyme. The results
suggest that Lys-319, which is distal to the active site, is important
to the flexibility/stability of the enzyme, as well as to its catalytic
machinery.
The enzymes belonging to the aspartic proteinases (EC 3.4.23.X) possess remarkable homology in their tertiary structures (Pitts et al., 1992). X-ray crystallographic data reveal that this group of enzymes shares a bilobal symmetry, with the two structural domains separated by a hinged substrate binding cleft. Two active site aspartic residues occupy the cleft to which admittance is restricted by a hinged flexible flap region (Suzuki et al., 1989). Although high in structural homology, these enzymes differ markedly in their catalytic properties, indicating that their activity is a result of subtle differences in protein structure.
Porcine pepsin (EC 3.4.23.1), a prominent member of the aspartic proteinases, was one of the first enzymes to be studied and the second to be crystallized (Northrop, 1930). Consequently, pepsin is the best understood of this family of proteinases and has been used as a model to study related enzymes (Abad-Zapatero et al., 1990; Lin et al., 1992). Pepsin is composed of 327 amino acid residues, has a calculated molecular mass of 34 kDa (Cooper et al., 1990), and is characterized by its low milk-clotting/proteolytic activity when compared with the milk-clotting enzyme of choice, chymosin (Suzuki et al., 1990). Previous investigations (Pearl and Blundell, 1984; Lin et al., 1989) into the functional activity of pepsin have been largely restricted to residues of the substrate binding pocket/active site due to their importance. This latter approach, however, assumes that the bulk of the protein structure plays a minor role in the functional activity of the enzyme, although mutations distal from the active site may influence enzymatic activity (Conrad, 1979). It has been suggested, recently, that enzymes behave more like mechanical devices than static biocatalysts implying areas external to the active site are vital to enzymatic activity (Williams, 1993).
It was the purpose of this study to observe the effect of Lys-319 mutations on the catalytic (proteolytic activity) and structural properties (changes in secondary structure, molecular modelling) of pepsin. Lys-319 is the sole lysine in the entire mature pepsin molecule. Located near the back side of the active cleft, Lys-319 is believed to have little interaction with prosegment residues during folding and is exposed to solvent in the mature enzyme (Cooper et al., 1990). It was hypothesized that the surface-protruding Lys residue plays a role in the catalysis and conformational stability/flexibility of the molecule.
The amino acid sequence of porcine pepsinogen, deduced from automated dideoxy terminator sequencing confirmed the sequence as reported by Lin et al.(1989) (data not shown). The molecular mass (34 kDa) and purity of the activated product, i.e. pepsin, were confirmed using SDS-polyacrylamide gel electrophoresis and Western blotting (data not shown).
Two mutations were introduced into the porcine pepsin gene using M13 in vitro mutagenesis as described by Kunkel(1985). The efficiency of the technique (75%) was such that screening was unnecessary. Confirmation of mutagenesis was accomplished by sequencing several plaques. The confirmed mutagenic 1.3-kb EcoRI fragments from M13 were subcloned back into the original pGBT-T19 vector. Restriction analysis, using SstI, of the newly constructed expression plasmids confirmed correct construction and orientation of the insert.
Refolding of expressed proteins followed the protocol of Lin et
al.(1989) and elution patterns similar to those described by these
authors for the various proteins on Sephacryl S-300 gel filtration were
observed (data not shown). FPLC anion exchange chromatography of the
various proteins indicated that the wild-type pepsinogen eluted at
approximately 15 min while Lys-319 Glu and Lys-319
Met
pepsinogens eluted at approximately 16 min (data not shown). The delay
in elution of the two mutant proteins was likely the result of the
increase in the net negative charge of the mutants interacting with the
anionite resin. The chromatographic data, however, would indicate that
the refolded structures of both mutants as well as the wild-type
protein were similar.
The hydrolytic and autolytic activity of
pepsin is reduced at pH 6, and its activity is fully regained upon
acidification (Ryle, 1966). However, at pH values above 6.0, the enzyme
is rapidly and irreversibly denatured (Cooper et al., 1990). A
downward shift in the upper pH limit of activity for Lys-319 Met
and Lys-319
Glu, using the synthetic peptide, to just below pH
4.0 (from pH 6.0 for the native pepsin) was observed (Fig. 1)
and suggests a decrease in the pH of alkaline denaturation. Further
investigation into the pH activity of the mutant proteins confirmed
that the inactivation pH had been shifted to approximately pH 4.0. Both
mutant proteins lost activity following incubation for 4 h at pH 5.3.
Alkaline denaturation of pepsin is reported to be a result of
irreversible denaturation in the N terminus domain (Lin et
al., 1993). Similarly, this decrease in pH activity from pH 6.0 to
4.0 for Lys-319
Met and Lys-319
Glu enzymes suggests that
the mutations to Lys-319 caused parts of the C-terminal domain to be
unstable similar to that observed in the N terminus by Lin et
al.(1993). Although there was a substantial decrease in the
inactivation pH for the mutants, there was no appreciable shift in the
pH optimum (Fig. 1). Wild-type pepsin exhibited a broad range of
optimum activity for this synthetic substrate, maintaining greater than
90% of its activity above pH 4.0. Both mutants displayed narrow optimum
pH values around pH 2 with the Lys-319
Glu mutant displaying
less than 20% activity at pH 1.4.
Figure 1:
Activity profiles at
varying pH using the synthetic substrate
Lys-Pro-Ala-Glu-Phe-Phe(NO)-Ala-Leu, for wild-type pepsin
(
), Lys-319
Met-pepsin (
), and Lys-319
Glu-pepsin (
). Experimental conditions are described under
``Experimental Procedures.''
The peptide,
Lys-Pro-Ala-Glu-Phe-(p-nitro)Phe-Ala-Leu, was chosen as the
substrate for the present study due to its high solubility
characteristics and excellent sensitivity for pepsin and other aspartic
proteinases (Fusek et al., 1990). The K values for cleavage of the synthetic octapeptide increased
in the order of wild-type, Lys-319
Met, Lys-319
Glu
pepsins (Table 1). As the K
values
increased, k
values for the mutant enzymes
decreased in the same order (wild-type pepsin, Lys-319
Met,
Lys-319
Glu) suggesting that the mutations have created general
conformational changes to the active site area. Furthermore, the ratio
of k
/K
decreased
in the order of wild-type pepsin, Lys-319
Met pepsin, and
finally Lys-319
Glu pepsin. Taken together, these results would
indicate that both the catalytic function and substrate recognition of
the pepsin molecule were affected by the mutations, the Lys-319
Glu mutation having a greater effect than the Lys-319
Met
mutation.
Supportive data to those observed for the kinetic
parameters and pH denaturation study were seen in the analysis of CD
spectral data for the various pepsins (Table 2). Comparison of
secondary structure fractions of the various pepsins at pH 2.1 and 5.3
indicated changes in -helix and
-sheet fractions, the
magnitude being more substantial for the mutant enzymes as compared to
the wild-type enzyme. Secondary structure fractions for the wild-type
pepsin were similar to those reported by previous researchers (James
and Sielecki, 1986; Yada and Nakai, 1986).
Differences in catalytic
parameters for the various pepsins observed at pH 2.1 (Table 1),
may have been a reflection of the proportion of -helix and
-sheet fractions where the Lys-319
Glu mutant had a higher
-helix and lower
-sheet content than either the wild-type or
Lys-319
Met pepsins. The
-sheet content of pepsin may be
responsible for the structural flexibility (or stability) given that
the CD (Yada and Nakai, 1986) and crystallographic (James and Sielecki,
1986; Abad-Zapatero et al., 1990) studies indicate that pepsin
is a
-sheet dominant protein. Examination of the
-sheet
contents from CD data indicated that the Lys-319
Glu and Lys-319
Met mutants exhibited a high degree of structural flexibility
which may have resulted in decreased enzymatic activities as compared
to the wild-type pepsin, i.e. the
-sheet contents of
these mutants, especially Lys-319
Glu, were much smaller than
that of the wild-type as calculated from crystallographic data
(approximately 40%). The substantial changes in
-helix and
-sheet fractions seen in the mutant enzymes, as a function of
increasing pH, corresponded to the absence of detectable enzymatic
activity at pH 5.3 (see Fig. 1). However, upon examination of
secondary structure fractions of the mutant proteins and the wild-type
pepsin at pH 5.3, only slight differences exist indicating that the
loss of enzymatic activity occurs with only minor changes in structure.
Although valuable structural information is obtained from the analysis
of CD data, no details regarding precise location of structural
fraction(s) or identification of areas of structural change are
obtained.
Recently, computer modelling/simulations have become a
valuable tool in yielding information as to the possible effects of
mutations on protein structure. The minimized structures of the Lys-319
Glu and Lys-319
Met pepsins were almost identical with
that of wild-type pepsin except for residues around the mutated lysine
residue. The total energies for the initial models of wild-type,
Lys-319
Glu, and Lys-319
Met pepsins were 10,508.6
kcal/mol, 10,522.0, and 10,492.3 which decreased to 2,960.94, 2,928.81,
and 2,954.63 after the energy minimizing calculations, respectively. In
the minimized models, root mean square deviations of main chains were
0.008 Å between the wild-type and Lys-319
Glu mutant and
0.007 Å between the wild-type and Lys-319
Met mutant.
These results suggested that the static structure of pepsin was not
affected by the replacement of Lys-319; however, flexibility of the
molecules may have been affected. Changes in flexibility may have
accounted for the changes seen in the above CD data and the enzyme
activity data of the mutant enzymes as compared to the wild-type
enzyme. To estimate the flexibility of the molecules, molecular
dynamics simulations on the minimized wild-type and mutant pepsin
models were applied. Distances of
-carbon at each 50 fs from the
initial position were calculated from the neighbor coordinate list
yielded from the simulations. The maximum values of the
-carbon
deviations were taken as an index of flexibility at each residue. The
flexibility indices are shown in Fig. 2. Differences in the
flexibility index among wild-type and mutants were plotted in order to
determine the effect of the mutation of Lys-319 on the flexibility of
the pepsin molecule (Fig. 3, A and B). As
suggested from the CD data, substantial effects on the flexibility
indices were observed in several sites which are suggested or assigned
as essential segments for catalysis. These sites include a flap loop
covering the active site, regions near the entrance of the active site
cleft, regions which experience large movement upon inhibitor binding
(subsite for peptide binding) (Chen et al., 1992), and
subdomains of the C-terminal domain. In pepsin, the flap loop is a very
flexible part of the enzyme. The indices of wild-type were around 1.5
to 4 Å throughout the loop. However, both mutants showed much
larger indices on the loop. In the Lys-319
Met mutant, the
largest index found in the molecule was observed in the loop. These
differences suggest that the loops in the mutants are bending in a
different manner from the loop in wild-type. Other large differences
among the mutants and the wild-type were observed around the two
helices in the N-terminal domain and the one loop in the C-terminal
domain, all of which are at the entrance of the active cleft. In
addition to these changes, larger or smaller values in the mutants than
the wild-type were observed in some subsite regions for peptide binding
(Chen et al., 1992). For example, Phe-117 in subsite S1 of
both mutants showed a smaller index than wild-type by -1.2
Å, and Gly-34 in S1 of Lys-319
Met showed a larger index
by 2.5 Å. The largest differences between the wild-type and
mutant proteins, however, were observed at subdomains of the C-terminal
domain. Abad-Zapatero et al.(1990) suggested that the
flexibility of these subdomains plays a structural role in mediating
substrate binding or determining the substrate specificity. Large
differences in the indices between the mutants and wild-type were
observed in the subdomain regions, i.e. from 3.0 Å at
Ile-252 of Lys-319
Glu mutant to -3.7 Å at Asp-242
of Lys-319
Met mutant. These differences observed on molecular
dynamics simulations suggest that the Lys-319 replacement resulted in
large effects on the structural flexibility which is essential for
effective catalysis.
Figure 2:
The maximum movement of -carbon
during the molecular dynamics simulations. Each line shows the maximum
movements (flexibility index) of
-carbons in each pepsin. Blue, red, and black are Lys-319
Glu,
Lys-319
Met, and wild-type enzymes, respectively. The flap loop
region, the substrate subsites, and the subdomains of the C-domain are
shown with a yellow background.
Figure 3:
The difference of the maximum movement at
each residue during the molecular dynamics simulations. The ribbon
diagram of pepsin models show the difference between wild-type and
each mutant pepsin, Lys-319 Glu (A) and Lys-319
Met (B). Red portions indicate that the maximum
movements (flexibility index) are larger than the counterpart of
wild-type. Skyblue portions indicate the opposite. Gray
portions mean both wild-type and mutant have similar indices. Thicker colored portions represent larger differences. Active
Asp-32 (left) and -215 (right) are in yellow, and the Glu and Met mutants are in purple.
It has been proposed that certain enzymes are mechanical devices rather than static biocatalysts, with similarity to a pump in processing substrate (Williams, 1993). This thought gives rationality to the bulk of an enzyme's structure, which is mostly thought of as incidental when considering merely the active site and subsite binding pockets, as the catalytic machinery. Pepsin has been shown to be extremely flexible in several areas including subdomains in the C-terminal domain (Abad-Zapatero et al., 1990) and the entrance region of the active cleft (Chen et al., 1992). It is proposed that the induced mutations in pepsin at Lys-319 disrupts the enzymatic activity by changing the flexibility of the entire protein molecule and, thereby, altering the catalytic activity of the enzyme.
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