From the
In order to analyze the structural requirement(s) for
proteolytic cleavage, synthetic oligopeptides corresponding to the
carboxyl-terminal (COOH-terminal) sequence of the precursor to the D1
protein (pD1) of the photosystem II reaction center, with or without
substituted side chain(s) around the cleavage site, were subjected to
enzymatic analysis with partially purified processing protease from
spinach. The efficiency of action as a competitive inhibitor of the
enzymatic cleavage of the COOH-terminal extension, as well as the
capacity to serve as a substrate, was used as an indication of
effective binding to the protease. Neither a COOH-terminal fragment
consisting of the 9 amino acids that are cleaved from pD1 by the
protease nor a COOH-terminal fragment of the mature protein consisting
of 15 amino acids inhibited the enzymatic processing of pD1. By
contrast, a COOH-terminal fragment of pD1 consisting of 24 amino acids,
which included the sequences of both the COOH-terminal extension and
the COOH-terminal 15 amino acids of the mature protein, was effective
both as a competitive inhibitor and as a substrate. This result
suggests that the structure formed by linkage between these two parts
of the protein moiety is important in the substrate-enzyme interaction.
Among substitutions around the cleavage site, the replacement of
Leu-343 by Ala (L343A) specifically destroyed the ability of the
oligopeptide to serve as either a substrate or an inhibitor, suggesting
that the presence of the hydrophobic Leu residue is crucial for the
formation of the recognition site. A series of six substitutions at
Ala-345 had marked effects on the value of V
The D1 protein is an integral component of the photosystem II
(PSII)(
The enzymatic
cleavage of the COOH-terminal extension of the precursor to the D1
protein (pD1) occurs on the carboxyl-terminal side (C-side) of Ala-344,
both in higher plants
(15, 16) and in the
cyanobacterium Synechocystis sp. PCC 6803
(12) . The
extension consists of 9 amino acids in higher plants and of 16 amino
acids in Synechocystis. To date, more than 45 psbA
genes for the D1 protein have been sequenced. These genes come from a
wide variety of organisms, from cyanobacteria to higher plants
(17, 18, 19) , but the deduced amino acid
sequences are highly homologous. One striking feature of the sequences
is that the amino acids upstream of the cleavage site are mostly
conserved, whereas the COOH-terminal extension, which is cleaved off by
the processing protease, is variable both in terms of its amino acid
sequence and its length. Although there are minor variations in the
upstream sequence, many of them appear to originate from only one gene,
represented by psbAI of Synechocystis sp. PCC 6803.
This gene is one of three psbA genes in this organism, but its
transcript has not yet been detected
(20) .
The protease
involved in the cleavage of the COOH-terminal extension of pD1 has been
extracted and purified from spinach, pea, and Scenedesmus, and
its molecular and the enzymatic properties have been analyzed
(21, 22, 23, 24) . A gene
( ctpA) that seems to correspond to this enzyme has recently
been identified in a cyanobacterium and sequenced
(25, 26) . However, details of the characteristics and
the mode of action of this enzyme have not been fully elucidated. For
example, the inhibitors of this protease are very different from those
of most proteases and the enzyme is now believed to be a new type of
protease
(23, 24) . Synthetic oligopeptides
corresponding to the COOH-terminal sequence of pD1 have been shown to
be efficient substrates for the enzymatic reaction
(27, 28) , and they have been employed in analysis of
the signals for recognition and binding
(28) . In a previous
study, synthetic oligopeptides of different lengths were subjected to
enzymatic analysis
(28) . The results suggested the importance
of chain length, as well as of the positive charge on Asp-342, for the
cleavage reaction by the enzyme in vitro(28) .
In
the present study, we synthesized oligopeptides that corresponded to
the deduced COOH-terminal sequence of pD1 of spinach, but we replaced
specific amino acid(s) around the cleavage site in order to analyze the
requirement for conserved amino acids in the enzymatic reaction and to
identify structural features in this region that are important for
recognition. The results of this analysis clearly demonstrate that
Leu-343 at the -2 position is essential for recognition of the
substrate by the enzyme. The amino acid side chain at the +1
position, by contrast, seems not to be important for recognition or
binding, but it does dramatically influence the rate of cleavage. It
appears that the secondary structure around the cleavage site, rather
than specific amino acids, is important for the proteolytic reaction.
Preparation of the Enzyme The partially purified COOH-terminal processing protease was prepared
from spinach thylakoids by the following procedure. Thylakoid membranes
for extraction of the enzyme were prepared from spinach leaves as
described elsewhere
(29) . The crude enzyme was prepared by
sonicating the thylakoid membranes (1 mg chlorophyll
ml
In the
experiment for which results are shown in Fig. 5, the inhibitory
effects of two of the substituted COOH-terminal oligopeptides described
above were analyzed in the cleavage reaction with S-24 as substrate.
The relationship between the reaction rate and the concentration is
shown by double-reciprocal plots of the data. As is clearly shown in
the figure, I346A was a typical competitive inhibition. By contrast,
the inhibition caused by L343A was weak and of a non-competitive
nature. These results confirm the conclusion that Leu at the 343th
position plays a crucial role in the recognition of the substrate by
the protease and they suggest that the structure of the substrate
formed as a result of the presence of this residue is important in the
interaction between the enzyme and its substrate.
Synthetic oligopeptides corresponding to the deduced
COOH-terminal sequence of pD1 can be used as substrates for the
processing protease, as shown in previous studies
(27, 28) . The affinity of these oligopeptides for the
processing enzyme can also be demonstrated by their inhibitory effects
on the cleavage of in vitro transcribed and translated
full-length pD1
(28) . The competitive nature of this inhibition
was demonstrated in the present study by using another synthetic
oligopeptide as substrate, as shown in Fig. 2. The COOH-terminal
27-mer with replacement of Asp-342 by Asn (S-27N) was a competitive
inhibitor of the enzymatic cleavage of the COOH-terminal 24-mer.
In
the experiment for which results are shown in Fig. 4, amino acid
side chain(s) around the cleavage site (C-side of Ala-344) were
selectively replaced by Ala to examine the sequence requirement for
precise cleavage of pD1. The results of our experiments indicated that
the cleavage was absolutely accurate and was restricted to the C-side
of Ala at the 344th position. The results also indicated that the
presence of a hydrophobic side chain at the 343rd position (Leu-343) is
crucial for enzymatic cleavage. A similar requirement has been reported
for cysteine-type proteases, such as papain and calpain
(32, 33) . For these proteases, although their
specificity is generally very wide, the presence of a hydrophobic
residue around the cleavage site in the substrate, rather than the
amino acids on either side of the cleavage site, is more important for
proteolysis
(32, 33) . One typical example is peptidase
A from Streptococcus. In its substrates, the amino acid
residues at the +1 and -1 positions are reported to be of
secondary importance, whereas a bulky uncharged side chain at the
-2 position is essential
(33) . Thus, the requirement for
substrate recognition by the enzyme seems to resemble that by the
COOH-terminal processing protease analyzed in this study. We can
speculate that the pD1-processing protease has a binding site near its
catalytic domain that interacts hydrophobically with Leu at the
-2 position of the substrate. However, the COOH-terminal
pD1-processing protease has been shown to be insensitive to all the
major classes of protease inhibitors, including those of cysteine-type
proteases. Thus, it can be categorized as a new type of protease with a
novel catalytic mechanism
(21, 22, 23, 24, 27, 28, 29) .
As shown in the previous study
(28) and in Figs. 2 and 3,
and also as generally expected, the affinity of the substrate for the
processing protease decreased dramatically when the substrate was
cleaved into the two products, namely the COOH-terminal extension and
the COOH-terminal moiety of the mature D1 protein. This result suggests
that the polypeptide bond at the cleavage site or the structure around
the cleavage site formed by the linkage between the two parts of the
molecule is important for the recognition, rather than the specific
amino acid side chain(s). It was reported in the previous paper
(28) that a COOH-terminal oligopeptide consisting of 11 amino
acids of pD1 was ineffective as a substrate, in spite of the fact that
Leu-343 and Asp-342, known to be important for recognition, were
present in the molecule. This earlier result also supports our
interpretation. In the previous report, it was shown that the
oligopeptide composed of the 16 COOH-terminal amino acids of pD1 (S-16)
had decreased effectiveness as a substrate when compared with longer
oligopeptides, perhaps because of insufficient chain length for the
formation of a normal recognition site
(28) .
In considering
the structure around the cleavage site, it is of interest to note that
there is a conserved Pro residue, which is generally considered to
break the helical structure of polypeptides, at the 340th position. The
function of the pD1-processing protease resembles that of signal
peptidases in prokaryotic and eukaryotic systems whose functional roles
have been examined in relation to the co-translational translocation of
secretory proteins
(34, 35) . Furthermore, the major
types of protease inhibitor have practically no effect on signal
peptidases. In such peptidases, a small uncharged amino acid is present
at the -1 position of the substrate and a Pro residue is usually
present from the -4 to the -6 positions. In signal
peptidases, it is believed that the presence of Pro in this region of
the substrate may be crucial for efficient presentation of the cleavage
site to the enzyme
(34) . If Pro-340 plays a similar role in
pD1, S-16 may have an insufficient
The kinetic analysis of
the reactions with COOH-terminal oligopeptides substituted at the
+1 position indicated that rank order of V
Nixon
and Diner
(12) genetically modified the D1 protein
( psbAIII gene) of Synechocystis sp. PCC 6803 by
site-directed mutagenesis at the +1 position relative to the site
of cleavage. They demonstrated that the processing was blocked when Ser
at the +1 position was changed to Pro (S345P). The result was
interpreted to indicate that the manganese cluster was unable to
assemble correctly in this mutant, which is deficient in photosynthesis
(12) . In our study, the Pro-substituted COOH-terminal
oligopeptide was not a potent inhibitor of the cleavage reaction (data
not shown), suggesting that the Ala-Pro bond is not only resistant to
the enzymatic cleavage but also causes an appreciable change in the
recognition signal. On the other hand, the S345A, S345R, and S345L
mutants of Synechocystis are able to grow photoautotrophically
and evolve oxygen at rates comparable to that of the wild-type strain
(12) . We have no corresponding data for these substitutions
in vitro. However, the calculated low probability of forming a
We reported previously that,
when the COOH-terminal processing activity was assayed using in
vitro transcribed and translated pD1 as substrate, the cleavage
reaction was completely inhibited by the presence of 50 mM
NaCl under our experimental conditions
(24) . The enzymatic
reaction with synthetic COOH-terminal oligopeptides as substrates, by
contrast, was much less sensitive to the ionic strength of the reaction
mixture.(
,
without affecting the binding affinity, as represented by
K
; the order of substitutions at residue
345 in terms of their effects on V
was Ala, Ser,
Phe, Cys > Gly > Val
Pro. With a Pro residue at position
345, the oligopeptide was practically inactive as a substrate.
)
reaction center
(1, 2) .
Together with its homologous counterpart, the D2 protein, it forms a
heterodimer, as in the reaction center of purple bacteria
(3) ,
providing binding sites for the pigments and redox cofactors that are
engaged in the primary and the secondary electron-transfer reactions
(4) . A Tyr residue on D1 also serves as the secondary electron
donor of the photosystem
(5) . An unusual property of this
essential subunit of the PSII reaction center is, however, the
extraordinarily high rate of its turnover in vivo(6, 7) , which is thought to represent the
damage-repair cycle of PSII
(8) . In this metabolic cycle, the
D1 subunit is synthesized by thylakoid-bound ribosomes on the stromal
surface of membranes as a precursor protein, which has a short
carboxyl-terminal (COOH-terminal) extension
(9, 10) .
This part of the protein is translocated immediately after synthesis
into the lumenal space and then it is excised enzymatically with a
half-time of several minutes, as clearly demonstrated by the results of
analysis by SDS-polyacrylamide gel electrophoresis of radiolabeled
proteins
(9) . As shown by previous studies of the LF-1 mutant
of Scenedesmus obliquues(11) and of genetically
engineered mutants of Synechocystis sp. PCC 6803
(12) ,
this COOH-terminal cleavage process is essential for the evolution of
oxygen in photosynthesis. In a study with Synechocystis, it
was proposed that a free carboxyl group at the COOH terminus of the D1
mature protein is, by itself, a ligand for the manganese cluster of the
oxygen-evolving complex
(12) . By contrast, genetically
truncated mutant proteins that lack the COOH-terminal extension, both
in Chlamydomonas reinhardtii(13) and in
Synechocystis sp. PCC 6803
(12) , as well as in
Euglena gracilis in which no extension is encoded by the
coding sequence for D1
(14) , have been shown to be able to
participate in photosynthesis, at least under the conditions tested to
date. Thus, the biological function(s) of the COOH-terminal extension
of the D1 protein is still obscure, although the cleavage process is
absolutely essential if such an extension is present.
) in 20 mM Tris-HCl (pH 7.2 at 25 °C)
buffer that contained 10 mM NaCl and 20 mM MgCl
with a sonicator (Sonifier 250; Branson, Danbury, CT) at 40 W at
a frequency of 20 kHz for 60 s at 0 °C, with subsequent
centrifugation at 188,000
g for 1 h. The resulting
supernatant was diluted with two volumes of 20 mM sodium
phosphate (pH 6.4 at 4 °C) buffer and then applied to a
hydroxylapatite column that had been pre-equilibrated with the same
buffer. The column was eluted with a linear gradient of 20-400
mM sodium phosphate. The active fraction from the
hydroxylapatite column was concentrated by ultrafiltration with an
Amicon YM-10 membrane (exclusion limit, M
=
10,000; Amicon, Beverly, MA), loaded onto a gel filtration column of
Sephadex G-75 (SF), which had been pre-equilibrated with 25 mM
HEPES-NaOH (pH 7.7 at 25 °C) buffer that contained 100 mM
NaCl and was then eluted with the same buffer at a flow rate of 4 ml
per h. The resultant partially purified enzyme contained no other
protease that cleaved pD1, as demonstrated by the absence of
nonspecific cleavage products even after prolonged incubation (see, for
example, Fig. 4). Substrate
Figure 4:
Profiles after HPLC of the products of
COOH-terminal cleavage of substituted synthetic oligopeptides by the
processing enzyme. Each oligopeptide was incubated with the partially
purified enzyme for 2.5 h at 25 °C. S, M-10,
P-9, and P`-9 indicate peaks of substrate
(COOH-terminal 19-mer of pD1 with or without substitution,
COOH-terminal 10-mer of mature D1 protein, COOH-terminal 9-mer of pD1,
and COOH-terminal 9-mer of substituted pD1 (Ile-346 to Ala)),
respectively.
Synthetic Oligopeptides
The substituted oligopeptides
corresponding to the COOH-terminal sequence of pD1, deduced from
psbA gene of spinach
(17) , were synthesized by a
peptide synthesizer (model 431A; Applied Biosystems, Foster City, CA)
and the protecting groups were removed by methods described by the
manufacturer. After elimination of impurities by reverse-phase, high
performance liquid chromatography (HPLC), the amino acid sequence of
each purified oligopeptide was confirmed with a protein sequencer
(model 477A, Applied Biosystems).
In Vitro Transcribed and Translated Precursor to the D1
Protein
pSPTB28, a pSP64 plasmid containing the psbA
gene from tobacco was provided by Dr. Sugiura (Nagoya University). The
pD1 protein was synthesized in a wheat germ cell-free translation
system (NEK-129 Z10; DuPont NEN) in the presence of
[S]methionine from mRNA generated by
transcription in vitro of EcoRI-linearized SPTB28 in
an SP6 system (Amersham International, Amersham, United Kingdom), in
accordance with the manufacturer's instructions
(29, 30) . Assay of Proteolytic Activity
Synthetic Oligopeptides as Substrates
The standard
assay mixture (46 µl) contained the indicated amount of substrate,
partially purified enzyme and 20 mM HEPES-NaOH (pH 7.7 at 4
°C) buffer. In the standard procedure, the reaction mixture was
incubated for a specified time at 25 °C and then the reaction was
terminated by addition of 9 µl of 18% (w/v) trichloroacetic acid.
The resultant mixture was centrifuged at 20,000 g for
20 min at ambient temperature (25 °C). The supernatant (50 µl)
was diluted with 130 µl of 0.1% (v/v) trifluoroacetic acid and 4
µl of 0.1 mM salicylic acid and passed through a Millipore
filter (pore size, 0.45 µm; Millipore, Bedford, MA). An aliquot of
the filtrate (170 µl) was analyzed by reverse-phase HPLC as
described in
(27, 28) .
In Vitro Transcribed and Translated Precursor to the D1
Protein as Substrate
The incubation mixture contained 2 µl
of in vitro transcribed and translated pD1 and 20 µl of a
solution of partially purified enzyme in 20 mM HEPES-NaOH (pH
7.7 at 25 °C) buffer. The reaction mixture was incubated at 25
°C for 2 h, and then the reaction was terminated by addition of an
equal volume of sample buffer for electrophoresis: 125 mM
Tris-HCl (pH 6.8 at 25 °C) buffer containing 4.6% (w/v) SDS, 20%
(w/v) glycerol, and 10% (v/v) 2-mercaptethanol. Proteins were separated
by SDS-polyacrylamide gel electrophoresis on gels that contained 6
M urea as described elsewhere
(31) , and then
radiolabeled proteins were detected by autoradiography. Analysis by HPLC The system consisted of an ``intelligent'' pump (type L-6210;
Hitachi, Tokyo), an integrator (type D-2500; Hitachi), an optical
detector (type L-4000UV; Hitachi) and a column oven (type L-5030;
Hitachi). A reverse-phase, prepacked stainless-steel column (4.6
250 mm, Shin-pack CLC-C8M; Shimadzu, Kyoto, Japan) equipped
with a guard column (Shin-pack G-C8
(4) ) was used at a flow rate
of 1.0 ml min
at 43 °C. Two solutions were used
for the elution: solution A, 0.1% (v/v) trifluoroacetic acid; and
solution B, 0.1% (v/v) trifluoroacetic acid in 70% (v/v) acetonitrile.
Elution was conducted with the following program: 1) 0-0.5 min,
solution A; 2) 0.5-20 min, increasing linear gradient from
solution A to 20% (v/v) solution B; 3) 20-40 min, increasing
linear gradient from the second step to 60% (v/v) solution B; 4)
40-46 min, decreasing linear gradient from the third step to
solution A. Elution was monitored at 220 nm. The integrated area
corresponding to the COOH-terminal 9 amino acids (9-mer) of pD1
analyzed by this system was estimated from data obtained with known
quantities of a standard 9-mer peptide, which was synthesized by
Kurabou Co. Ltd. (Neyagawa, Japan).
Inhibitory Effects of COOH-terminal
Oligopeptides
As described in the previous study
(28) ,
synthetic oligopeptides corresponding to the deduced COOH-terminal
sequence of pD1 of spinach (Fig. 1), which can be used as the
substrate for the isolated COOH-terminal processing protease, also have
an inhibitory effect on the enzymatic reaction with in vitro transcribed and translated full-length pD1 as substrate. The
inhibition of COOH-terminal cleavage by the addition of the
COOH-terminal oligopeptides can also be demonstrated for the cleavage
reaction when COOH-terminal oligopeptides are used as substrate, as
shown in Fig. 2. For example, the enzymatic COOH-terminal
cleavage of a 24-mer (COOH-terminal 24 amino acids sequence of pD1;
S-24) is inhibited by the addition of a 27-mer with substitution of
Asp-342 by Asn (S-27N). The result suggests that S-27N still has
affinity for the COOH-terminal processing protease, although its
efficiency as a substrate is only about one-fifth of that of
non-substituted 27-mer (S-27) (data not shown; see Ref. 28).
Figure 1:
The
carboxyl-terminal sequence of the protein to the D1 precursor, as
deduced from the nucleotide sequence of the psbA gene of
spinach. Numbering refers to number of residues from the NH terminus (Met-1). The conserved residues are indicated by
dots. The enzymatic cleavage occurs on the carboxyl side of
Ala-344 ( arrow).
Figure 2:
Lineweaver-Burk plots of reaction rates
versus concentrations of substrate. The substrate (S-24,
COOH-terminal 24-mer) was incubated with partially purified enzyme for
5 h at 25 °C, in the presence of 0 ( closedcircles), 417 ( opencircles), and 626
or 645 ( closedtriangles) µM synthetic
oligopeptide as indicated: a, S-27N, COOH-terminal
oligopeptide (27-mer) corresponding to the D1 precursor of spinach,
with replacement of Asp-342 by Asn; b, M-15, COOH-terminal
oligopeptide (15-mer) corresponding to the mature D1 protein of
spinach. The rate of the enzymatic reaction ( V) is shown in
relative units (nmol of 9-mer produced/h) based on quantification of
the cleaved product by HPLC and known quantities of synthetic standard
peptide (9-mer).
In
order to understand the mechanism of inhibition by the substituted
COOH-terminal oligopeptide (S-27N), the enzymatic reaction with S-24 as
substrate was kinetically analyzed in Fig. 2 a. The
concentration of S-27N in the reaction mixture was varied from 0 to 626
mM. Using the initial rate of COOH-terminal cleavage at 25
°C, as determined by HPLC, we plotted the concentration of
substrate versus the reaction rate as described by Lineweaver
and Burk. The plot clearly indicated that inhibition by S-27N was
competitive. Thus, S-27N bound to the enzyme at the same site as the
substrate (S-24), although the cleavage reaction was not efficient,
perhaps because of the absence of a negative charge at the 342th
position, as discussed elsewhere
(28) . Reflecting this result,
similar inhibition of the COOH-terminal cleavage of in vitro transcribed and translated radiolabeled pD1 was observed upon the
addition of S-24 or S-27N to the reaction mixture, as shown in
Fig. 3
.
Figure 3:
Inhibitory effects of synthetic
oligopeptides on the processing of in vitro translated D1
precursor protein (pD1) as substrate. The partially purified processing
enzyme and substrate (pD1) were incubated for 2 h at 25 °C in the
presence of each synthetic oligopeptide at 1.09 mM.
C, no addition; 1, COOH-terminal 9-mer, COOH-terminal
extension (P-9); 2, COOH-terminal 24-mer
(S-24); 3, COOH-terminal 27-mer with Asp-342 replaced
by Asn (S-27N).
In order to analyze the structural requirement(s) for
the substrate in its interaction with the COOH-terminal processing
enzyme, we examined the affinity for the enzyme of the COOH-terminal
extension of 9 amino acids by itself (P-9), and that of the
COOH-terminal fragment of the mature protein by analyzing their
inhibitory effects on the enzymatic reaction with either pD1 or the
COOH-terminal oligopeptide as substrate. As shown in previous studies
(28) and also in Fig. 3, the COOH-terminal extension of 9
amino acids (P-9) has, by itself, no effect on the COOH-terminal
cleavage of pD1 by the isolated protease. The oligopeptide
corresponding to the sequence of 15 COOH-terminal amino acids of the
mature protein (N-15) was also ineffective as an inhibitor in the
reaction with S-24 as substrate, over a wide range of concentrations
(Fig. 2 b). From this analysis, we concluded that both
the COOH-terminal extension, which is cleaved off by the processing
protease, and the well-conserved COOH-terminal sequence of the mature
D1 protein have practically no affinity for the enzyme and, thus, it is
the structure formed by the linkage between these two parts of the
protein that appears to be important in the substrate-enzyme
interaction. This conclusion is, in fact, rather general: the substrate
usually has high affinity for the enzyme while the reaction product(s)
usually has low affinity.
Substitution of Amino Acids around the Cleavage
Site
Since results obtained in the preceding section indicated
that the structure around the cleavage site of the substrate is
important for recognition and binding, oligopeptides corresponding to
the 19 COOH-terminal amino acids of the precursor to the D1 protein
with specific amino acid substitution(s) around the cleavage site were
synthesized and subjected to enzymatic analysis. In one oligopeptide,
Leu-343, which is completely conserved in all the deduced sequences of
D1 proteins reported to date, was replaced by Ala (L343A); in the
second oligopeptide, Ile-346, which is not always conserved but is
present in many cases in the sequence of pD1, was replaced by Ala
(I346A); and in the third, both Leu-343 and Ile-346 were replaced by
Ala so as to yield a sequence of 4 successive Ala residues between
positions 343 and 346th (L343A and I346A). When each substrate was
incubated with the partially purified enzyme, no cleavage product was
formed with L343A and with L343A and I346A within a 24-h incubation
(Fig. 4). By contrast, when I346A was used as substrate, the
enzyme cleaved the polypeptide at the normal site, namely on the C-side
of Ala-344, as shown by amino acid sequence analysis of the product
that was detected by HPLC (data not shown). Kinetic analysis of the
cleavage reaction with I346A yielded a Kof about 300 µM, similar to the value for the
control substrate (S-24), whereas V
was a little
lower than that for S-24, suggesting that this substituted oligopeptide
has affinity for the enzyme similar to that of the control substrate
but that there is a decrease in the efficiency of turnover for unknown
reasons. These results clearly indicate that the processing enzyme
specifically recognizes the C-side of Ala-344 and, more importantly,
that Leu-343 is an essential residue for the binding, possibly
generating the intrinsic structure necessary for recognition and/or
cleavage of the substrate by the processing enzyme.
Figure 5:
Lineweaver-Burk plots of reaction rates
versus substrate concentrations. The substrate (S-19,
COOH-terminal 19-mer) was incubated for 2.5 h at 25 °C in the
absence ( opencircles) or presence ( closedcircles or triangles) of substituted
COOH-terminal oligopeptides as indicated.
Substitution at the Cleavage Site (+1
Position)
The site of cleavage of its substrate by the
pD1-processing protease is on the C-side of Ala-344, as mentioned above
(15, 16) . Ala at the +1 position in the
COOH-terminal extension is relatively highly conserved among species.
However, in some cyanobacteria and in C. reinhardtii, this
amino acid is replaced by Ser. In order to analyze the significance of
this residue, the amino acid at the +1 position was replaced by
Val, Gly, Cys, Phe, Ser, or Pro and cleavage reactions with these
substrates were analyzed. Fig. 6 a shows the time course
of COOH-terminal cleavage by the isolated processing protease of the
six oligopeptides with a substitution at the +1 position. The
COOH-terminal cleavage was completely prevented when Ala-345 was
replaced by Pro (A345P), even after prolonged incubation (24 h),
reflecting data obtained by analysis in vivo(12) . The
Cys-substituted substrate (A345C) had a similar initial rate of
cleavage ( V) to the control (S-19; Ala at the
+1 position) during incubation for less than 2 h. Unusual behavior
was observed, however, after longer incubation, perhaps due to
interactions between accumulated reaction products that contained
NH
-terminal Cys residues. Substitution by Val (A345V) and
by Gly (A345G) decreased the initial rate, whereas substitutions by Ser
and Phe (A345S, A345F) gave similar kinetics to the control. In order
to analyze the effects of these substitutions on the enzyme kinetics,
values of K
and V
were estimated from double-reciprocal plots of the kinetic data
(Fig. 6 b), as summarized in . The
V
value for the oligopeptide with Val at
position 345 was about 30% and that with Gly was about 50% of the
control value. By contrast, the K
values
for all these substituted oligopeptides, except for A345P, were about
300 µM, being nearly the same value as that of the
control. Thus, efficiency of cleavage can be summarized, in terms of
the amino acid at position 345, as follows: Phe, Ala, Ser, Cys > Gly
> Val
Pro. Since the K
is related
to the dissociation constant of the enzyme-substrate complex, it can be
concluded that the affinity of binding of the substrate to the enzyme
was not appreciably affected by these substitutions at the +1
position, although the rate of cleavage was very seriously affected. We
also replaced Glu-347 by Gln (E347Q), since a negative charge (Glu or
Asp) at this position is relatively highly conserved among species and
a substrate with two negative charges in symmetrical positions on
either side of the cleavage site could be imagined to be important for
recognition. However, this substitution did not influence the rate of
cleavage or the K
, as shown in
.
Figure 6:
Kinetic analysis of COOH-terminal cleavage
of oligopeptides substituted at the +1 position. Time courses
( a) and Lineweaver-Burk plots ( b) of reaction rates
versus substrate concentrations, for reactions with +1
substituted COOH-terminal oligopeptides. The enzymatic reactions were
carried out at 25 °C.
-turn structure because of its
short chain-length, with resultant slow cleavage, as shown in the
previous study
(28) . Based on the similarities between the
substrates of these two types of protease, we can speculate that the
intrinsic structure formed by Leu-343 in the pD1 protein, perhaps in
collaboration with Asp-342 and Pro-340 contributes to the signal that
is recognized by the processing protease.
for the cleavage reaction was: Ala, Phe, Ser, Cys > Gly >
Val
Pro. This order disagrees with the predictions of von
Heijne's model
(36) , in which it is proposed that small
residues are associated with higher rates in the cleavage of signal
peptides. However, the average probability values for the secondary
structure around the cleavage site in the COOH-terminal extension for
each +1 substituted oligopeptide of 38 amino acids, calculated by
the GOR predictive method
(37) , indicated that the order of
probable values of a
-extended structure is Val > Gly > Cys,
Ser, Phe > Ala. This order is roughly consistent with the reverse of
the order for the COOH-terminal cleavage of substituted oligopeptides
in this study, suggesting that a moderate
-helical structure is
necessary for presentation of the cleavage site to the enzyme.
-extended structure for the Arg and Leu substitutions is
consistent with the interpretation discussed above, namely that a
moderate
-helical structure in the substrate is necessary for
proper presentation of the cleavage site to the enzyme. Kinetic
analysis indicated that the +1 substitution does not influence the
K
value, although it greatly influences
the V
value. Thus, this position is probably not
important for binding to the enzyme.
)
This result suggests the possibility
that the mechanism of substrate recognition differens to some extent
between the two assay systems that use either a small oligopeptide or
full-length pD1, respectively. A relatively higher value of
K
of about 300 µM and an
apparent low turnover number, as estimated for the catalytic reaction
with synthetic oligopeptides as substrates, might be related to the
size of the substrate. Thus, in the catalytic reaction with pD1 as
substrate, or in vivo, some charged residues situated upstream
of the sequence, for example, on the lumenal extrusions between helices
I and II or helices III and IV, might also contribute to the binding
and stabilization of the enzyme-pD1 complex. These possibilities will
be investigated in forthcoming analyses both in vivo and
in vitro.
Table: Kinetic data for the C-terminal cleavage of
substituted oligopeptides
©1995 by The American Society for Biochemistry and Molecular Biology, Inc.