ABSTRACT
We have recently demonstrated that synthetic peptides modeled on
the extension peptide of malate dehydrogenase can be a good substrate
of mitochondrial processing peptidase and that arginine residues
present at positions -2 or -3 and distant from the cleavage
point were important for recognition by the enzyme (Niidome, T.,
Kitada, S., Shimokata, K., Ogishima, T., and Ito, A.(1994) J. Biol.
Chem. 269, 24719-24722). We further investigated the
elements required for substrates of the protease. To analyze the
reaction by a more rapid yet quantitative method, we have developed
intramolecularly quenched fluorescent substrates. Using the fluorogenic
substrates we demonstrated that at least one of the proline and glycine
between the distal and proximal arginine residues was also important
while other connecting sequences were dispensable. In addition, the
protease showed considerable preference for aromatic and, to a lesser
extent, hydrophobic amino acids in the P
`-position. These
results together with the previous data suggest that the proximal and
distal arginine residues, proline and/or glycine between them, and
P
` amino acid could be critical determinants for the
specific cleavage of the substrates by the protease.
Most mitochondrial proteins are synthesized on cytoplasmic
ribosomes and transported into their correct mitochondrial component.
The majority of them carry N-terminal extension peptides that target
the protein molecules to the organelle. Mitochondrial processing
peptidase (MPP) (
)is localized in the mitochondrial matrix
and is responsible for proteolytic cleavage of the extension peptides
after or during the transportation(1, 2, 3) .
The enzyme is a metalloprotease and forms a heterodimer consisting of
structurally related
- and
-subunits(4, 5, 6, 7, 8, 9, 10, 11) .
We have recently demonstrated that the
-subunit is a catalytic
one(12) . MPP acts exclusively on the precursor forms of
mitochondrial proteins, whose extension peptides are heterogeneous in
sequence(13, 14, 15) . The absence of
apparent sequence homologies raises a question about how MPP
specifically recognizes the extension peptides and cleaves them at a
single site. Attempts have been made to solve the question, mainly by
use of in vitro translated and radiolabeled mutants of
mitochondrial precursors. By detecting mature forms from the in
vitro translated precursors on SDS-polyacrylamide gel
electrophoresis followed by fluorography, the processing activity has
so far been evaluated. Those studies pointed out the importance of
basic amino acids near the C terminus and neutral amino acids in the
middle portion of the extension
peptides(16, 17, 18, 19, 20) .
The method, however, is time-consuming, and the obtained data are not
quantitative. Thus the conventional method is unsuitable for kinetic
analysis of the reaction. To overcome the limitation of the assay
method, we developed a new method(21) , which employed as the
substrate synthetic peptides that were modeled on the extension peptide
of rat malate dehydrogenase. This method enabled us to reveal the
importance of arginine residues that are located at the -2 or
-3 position and at a position distant from the cleavage point.
To further investigate the structural elements in the substrates
required for recognition by MPP, we developed new substrates that are
based on intramolecular quenching of fluorescence. The fluorescent
peptides had a 2-aminobenzoic acid fluorophore at the N-terminal end
and a 3-nitrotyrosine quencher at a C-terminal portion from the
scissile bond in the peptides modeled on the malate dehydrogenase
extension peptide(22) . They were efficiently cleaved if they
contained the distal and proximal arginine residues. The results from
kinetic studies using the substrates revealed that proline and/or
glycine residues between the critical arginine residues were also
important while other connecting sequences were dispensable. They also
indicated that the enzyme exhibited considerable preference for
aromatic and, to a lesser extent, hydrophobic amino acids at the
P
`-position. (
)
EXPERIMENTAL PROCEDURES
Preparation of Peptides
The fluorogenic
substrates contained 2aminobenzoic acid (ABz) as a fluorescent group at
the N-terminal end and 3-nitrotyrosine (Tyr(NO
)) as a
quenching group between the MPP cleavage site and the C-terminal end.
We synthesized the peptides manually in solid phase employing N-
-9-fluorenylmethoxycarbonyl (Fmoc) strategy (23) by in situ coupling
method(24, 25, 26) . The following Fmoc amino
acid derivatives were used:
Fmoc-(N-
-methoxy-2,3,6-trimethylbenzensulfonyl)Arg,
Fmoc-(N-
-trytyl)Asn, Fmoc-(N-
-trytyl)Gln,
Fmoc-(O-t-butyl)Ser,
Fmoc-(O-t-butyl)Thr, and
Fmoc-(O-t-butyl)Tyr. Other Fmoc amino acids including
Fmoc-3-nitrotyrosine were used without side-chain protection. The
completed peptides were deprotected and cleaved from the resin with
trifluoroacetic acid/trimethylsilyl bromide as described elsewhere (27) . The peptides were purified by preparative reversed-phase
high performance liquid chromatography (HPLC), and their purity was
confirmed by amino acid analysis. The fluorogenic substrates that had
various amino acid residues at P
` were synthesized by a
fragment condensation method. Briefly, a series of XSTY(NO
)AQNN peptides was synthesized on p-alkoxybenzyl-alcohol-polystyrene resin as a C-terminal half
of the substrates. Here, X was F, Y, L, V, A, G, S, or R.
Fmoc-Gly was attached to 2-chlorotritylchloride resin (28) as
the P
amino acid, and the N-terminal counterpart was
synthesized on the resin. After completion, the side-chain protected
peptides were cleaved from the resin with acetic
acid/trifluoroethanol/dichloromethane (1:2:7) as previously described (29) . The side-chain protected peptide was coupled with the XSTY(NO
)AQNN-p-alkoxybenzyl-alcohol-polystyrene
resin. The condensed peptides were deprotected and cleaved from the
resin as described above. Peptides for inhibition experiment were
synthesized as peptide amides on
trialkoxy-diphenyl-aminomethyl-polystyrene resin(30) .
Analytical Methods
Fluorescence was measured at 25
°C on a Hitachi F-2000 fluorescence spectrophotometer. Excitation
was at 315 nm, and emission was measured at 420 nm. The peptide
concentrations were determined from the final fluorescence (F
) after cleavage of the peptides with trypsin
or chymotrypsin using ABz-Gly-NH
as a standard. The
standard was synthesized as described elsewhere(22) .
Concentrations of peptides were determined by the ninhydrin method (31) after the peptides were hydrolyzed in 2.5 N NaOH
at 100 °C for 90 min.
Enzyme Assay
The reaction mixture contained 30
nM substrate and 0.05-0.1 pmol of purified MPP in 1 ml
of 20 mM HEPES-NaOH buffer (pH 7.6). The reaction was started
by addition of the enzyme. Increase of the fluorescence intensity was
monitored, and the initial velocities, which had been corrected for the
initial fluorescence (F
) of the substrates, were
obtained. The k
/K
values
were determined from the equation, v = (k
/K
)[E][S],
where [E] and [S] are enzyme and substrate
concentrations, respectively. This equation is valid when the substrate
concentrations are much lower than K
.
Inhibitory Effect of Synthetic Peptides on the Reaction
with Fluorogenic Substrates
The processing reaction was analyzed
in a total volume of 1 ml. The substrate was 30 pmol of
ABz-LARPVGAALRRSFSTY(NO
)AQNN, and inhibitors were the
peptides at various concentrations that consisted of partial sequences
of the malate dehydrogenase extension peptide.
Analysis of Reaction Products
The enzyme reaction
was performed with 0.5 mM of the substrates and 0.2 pmol of
purified MPP in 0.2 ml of 20 mM HEPES-NaOH buffer (pH 7.6) for
30 min at 25 °C. The reaction was terminated by addition of 0.2 ml
of acetonitrile and 10 µl of 10% trifluoroacetic acid. After
centrifugation at 10,000
g for 5 min, the supernatant
was applied to a Cosmosil 5C18-AR column (10
250 mm; Nacalai
Tesque, Kyoto, Japan) equilibrated with 1% acetonitrile containing 0.1%
trifluoroacetic acid. The products were separated by a linear gradient
of acetonitrile (1-51%, 30 min) and collected. The separated
products were analyzed by amino acid analysis.
Materials
MPP was purified from bovine or rat
liver mitochondria according to the method of Ou et
al.(6) . Solvents for peptide synthesis were purchased
from Kanto Chemical Co. Ltd. (Tokyo). 2-Chlorotritylchloride resin was
from Novabiochem (Läufelfingen, Switzerland). Other
reagents for peptide synthesis were from Watanabe Chemical Industries,
Ltd. (Hiroshima, Japan). Fmoc-Tyr(NO
) was prepared by
coupling of Tyr(NO
) (Janssen Chimica, Geel, Belgium) with
Fmoc-N-succinimidyl carbonate as previously
described(32) , and the di-Fmoc derivative was removed
on Wakogel C-100 (Wako Pure Chemical Industries, Osaka, Japan).
RESULTS AND DISCUSSION
Design of Fluorogenic Substrates
Fig. 1shows the sequence of the extension peptide of rat
malate dehydrogenase. Consisting of 24 amino acids, the peptide has an
MPP cleavage site between Ser and Phe and a mitochondrial intermediate
peptidase cleavage site between Asn and Ala(14, 33) .
The fluorescent donor ABz and acceptor Tyr(NO
) were
introduced at the N-terminal end and at a position C-terminal to the
scissile bond, respectively, in the extension peptide. We first
synthesized a derivative of the full-length malate dehydrogenase
extension peptide, where Tyr(NO
) was introduced at the 20th
position (numbering from the original N terminus in pre-malate
dehydrogenase), and ABz was connected to Met
. Fluorescence
increased upon addition of MPP, and the increase continued linearly, at
least for the first 2-3 min. Addition of EDTA-Na at a final
concentration of 0.1-10 mM inhibited the increase of
fluorescence (Fig. 2) as had been observed in processing of both in vitro synthesized precursors (6) and nonfluorogenic
synthetic peptides(21) . The inhibition was not rapid,
indicating that the metal binds tightly at the active pocket of MPP.
Figure 1:
Amino acid sequence of rat
malate dehydrogenase precursor. The arrows indicate the first
and second cleavage sites by MPP and mitochondrial intermediate
peptidase (MIP), respectively.
Figure 2:
Increase of fluorescence on the cleavage
of ABzL5Y*20 and its inhibition by EDTA. The reaction was started by
addition of the enzyme solution to a cuvette containing various
concentrations of EDTA-Na. Substrate was
ABz-MLSALARPVGAALRRSFSTY(NO
)AQNN. Arrows indicate
addition of the enzyme. See ``Experimental Procedures'' for
details.
Although the fluorogenic peptide of full-length was efficiently
cleaved, it showed initially about 50% of the fluorescence of the fully
cleaved form. A substrate with such a high initial fluorescence has a
limited increase of fluorescence, which lowers the sensitivity of the
detection. Since long range resonance energy transfer is considered to
operate in this quenching(22) , the efficiency should improve
as distance between the donor and acceptor is reduced. Thus, we
synthesized a series of N-terminally truncated substrates to obtain
well quenched and yet efficiently cleaved substrates. Initial
fluorescence intensity of the peptides decreased, i.e. the
quenching efficiencies improved as the distance between the
chromophores was reduced (Table 1). MPP cleaved the peptides that
started from Ser
and Leu
as efficiently as that
from Met
. It also cleaved the peptides that started from
Arg
a little slower. The cleavage rates dropped
dramatically for the peptides that lacked the sequences from Met
to Arg
. Here, we could reconfirm the importance of
the distal arginine for substrate recognition as we have previously
shown by using the nonfluorogenic substrates(21) .
We also
synthesized peptides that had the Tyr(NO
) acceptor at the
17th (P
`) or 18th (P
`) position. The peptide
that had the acceptor at the P
` position (peptide 8 in Table 1) had a drastically reduced cleavage efficiency compared
to the peptide that had the acceptor at the 20th (peptide 1 in Table 1). Introduction of Tyr(NO
) into the
P
`-position significantly elevated the cleavage efficiency
when compared with the peptide having the acceptor at the
P
`-position but not when compared with the peptide having
the acceptor at the 20th position (compare peptides 9 and 3 in Table 1). Since the substrates that connected ABz to
Met
, Ser
, and Leu
were cleaved at
nearly the same rate, we hereafter used
ABz-LARPVGAALRRSFSTY(NO
)AQNN as a typical substrate
designating it as ABzL5Y*20.
Identification of the Cleavage Site in the Peptides That
Reacted with MPP
The reaction products from the substrates that
connected ABz to Met
, Leu
, and Arg
were separated by HPLC. Fig. 3shows that each
chromatogram gave only three components when monitored at 215 nm. The
peptide eluted latest corresponded to the substrate. The earliest
eluted fractions had an identical retention time about 22.0 min. The
first and third fractions showed an absorbance at 280 nm. They should
contain Tyr(NO
) since it has an absorption maximum at 276
nm at pH 2.0. Amino acid analysis of the fractions revealed that the
peptide with retention time of 22.0 was FSTY(NO
)AQNN and
that the peptides of the second peak were the N-terminal counterpart.
Other fragments than those peptides did not appear after a prolonged
incubation with MPP (data not shown). These results demonstrate that
the substrates were cleaved at the correct site by MPP.
Figure 3:
Identification of the reaction products by
HPLC. The substrates were reacted with MPP, and the reaction products
as well as the substrates were applied to a reversed-phase HPLC. The
substrates were: A,
ABz-MLSALARPVGAALRRSFSTY(NO
)AQNN; B,
ABz-LARPVGAALRRSFSTY(NO
)AQNN; and C,
ABz-RPVGAALRRSFSTY(NO
)AQNN. The peak fractions were
collected and analyzed for amino acid composition. See
``Experimental Procedures'' for
details.
Deletion and Replacement of the Intervening Sequences
between the Proximal and Distal Arginine Residues
The arginine
residue that is located at the -2 or -3 position from the
scissile bond in the extension peptide of malate dehydrogenase together
with the basic amino acid residue at a position distant therefrom is
important for recognition by MPP as previously shown(21) . We
then analyzed the role of the intervening sequences between the
proximal and distal arginine residues (Table 2). Cleavage
efficiencies of the substrates that lacked Ala
,
Ala
-Ala
, and
Ala
-Ala
-Leu
were
essentially the same as ABzL5Y*20. The efficiency dramatically reduced,
however, when deletion extended to Arg
. This seemed to be
discrepant with our previous result that alanine was replaceable for
Arg
(21) . Although we are unable to clearly
explain the discrepancy right now this arginine could serve only as a
spacer to give an appropriate distance between the proximal
(Arg
) and distal (Arg
) arginine residues.
Replacement of Pro
with Gly did not significantly alter the
cleavage efficiency of the peptide. However, replacement of Pro
and Gly
with Ala reduced the cleavage efficiency. In
a separate experiment using the synthetic peptide/HPLC assay system, we
have also obtained a similar result that MPP cleaved
MLSALARAVAAALRRSFSTSA less efficiently than MLSALARPVGAALRRSFSTSA with K
values of 6.0 µM (versus 0.7 µM) without significant change of V
values (5.0 versus 8.4 pmol/min). (
)These results indicate that the sequence from Ala
to Leu
was dispensable while at least one of the
-helix-breaking amino acid residues (Pro
and
Gly
) was important.
Inhibitory Effect of Synthetic Peptides That Have Partial
Sequences of the Malate Dehydrogenase Extension Peptide
We
measured the processing reaction toward the fluorogenic substrate
(ABzL5Y*20) in the presence of synthetic peptides to elucidate the
structural or sequential elements responsible to the specific
recognition by MPP. We first synthesized six kinds of peptides with
five consecutive sequences of the malate dehydrogenase extension
peptide (LARPV-NH
, PVGAA-NH
,
AALRR-NH
, RRSFS-NH
, FSTSA-NH
,
SAQNN-NH
) that corresponded to the amino acid sequences
from Leu
to Asn
. As a control, a synthetic
peptide corresponding to the N-terminal 13 amino acid residues of
mature malate dehydrogenase did not inhibit the reaction even at 0.1
mM (data not shown). None of the six peptides had any effect
on the cleavage of ABzL5Y*20 even at 0.1 mM (data not shown
except FSTSA-NH
). We then synthesized longer peptides that
elongated N-terminally from FSTSA-NH
and examined their
inhibitory potency (Fig. 4). The peptides connected with Ser and
Arg-Ser inhibited the reaction by 10 and 20%, respectively, at 2
µM. Inhibition was enhanced when Arg-Arg-Ser was connected
to FSTSA-NH
. Further elongation of RRSFSTSA-NH
with Ala gave only slight enhancement of the inhibitory effect.
The peptides connected with Ala, Ala-Ala, and Ala-Ala-Ala-Ala-Ala
inhibited the processing reaction to essentially the same extent.
Introduction of arginine to AAAAARRSFSTSA-NH
at a position
that corresponded to the distal position of the substrate, i.e. ARAAARRSFSTSA-NH
, greatly elevated the inhibitory
potency of the peptide. Further inhibition was observed (50% inhibition
at 20 nM) by introduction of a PVG sequence between the
provisional proximal and distal arginine residues
(ARPVGRRSFSTSA-NH
). Among the peptides above, at least
ARAAARRSFSTSA-NH
and ARPVGRRSFSTSA-NH
could act
as substrates of MPP (data not shown).
Figure 4:
Inhibitory effects of synthetic peptides
that have partial sequences of the malate dehydrogenase extension
peptide. The reaction toward ABzL5Y*20 was measured in the presence of
various concentrations of the synthetic peptides. The remaining
activity was determined and is given as percentage of the activity in
the absence of added peptide. See ``Experimental Procedures''
for details.
Recent NMR studies (34, 35) have indicated the role of proline and
glycine in mitochondrial extension peptides. They break a continuous
-helix from the middle portion of the extension peptides to the
cleavage point making the precursors competent for processing. Our
result also supported importance of the proline and glycine residues in
the extension peptide. The result of the present inhibition experiment
indicates that binding ability of synthetic peptides to MPP improves if
the
-helix-breaking amino acids are present between the
provisional proximal and distal arginine residues. Tight binding of the
peptides having a PVG sequence together with the finding that
replacement of the proline and glycine with alanine lead to significant
decrease of cleavage efficiency of the peptide (Table 2) supports
importance of these amino acids.
Preference for Amino Acids at the
P
`-position
The resistance to cleavage of the
peptide having Tyr(NO
) at P
` (Table 1)
suggested the limited acceptance of P
` residues in the
substrate by MPP. To confirm this, we synthesized a series of peptides
that had varied amino acids at the P
`-position. For
synthetic convenience, the P
amino acid was replaced by Gly
for Ser in the peptides. This conversion resulted in about half
reduction of the cleavage efficiency. Fig. 5shows the cleavage
efficiencies of the peptides having altered P
` amino acids.
Alteration of Phe to Tyr reduced the efficiency to about 50%, and that
to Leu and Ala further reduced the efficiencies to about 10%. Cleavage
efficiencies of the other peptides were below the detection limit.
These results demonstrate that MPP exhibits marked preference for
aromatic amino acids and, to a lesser extent, hydrophobic amino acids
at the P
`-position. Such amino acid residues are abundant
at P
` at least for the mitochondrial precursors that
receive two-step cleavage(14) . Although this position is
considered to be one of the recognition sites for the second processing
by mitochondrial intermediate peptidase(36, 37) ,
Arretz et al.(19) have recently reported importance
of P
` for Neurospora MPP. They mutated
phenylalanine at the P
` to lysine in a chimeric precursor
of cytochrome b
and found that the mutated
precursor did not undergo processing by purified MPP. We are not sure
right now if preference for aromatic and hydrophobic amino acids at the
P
`-position could also be applicable to the substrates that
undergo one-step cleavage by MPP. At least for the precursors receiving
two-step cleavage, the P
` amino acid together with the
proximal, distal, and flexible amino acids could determine the
substrate specificity and cleavage position of MPP.
Figure 5:
Preference of MPP for amino acids at the
P
`-position. Derivatives of ABzL5Y*20 that have Gly at
P
(originally Pro) and various amino acid residues at
P
were assayed. The activity toward
ABz-LARPVGAALRRGFSTY(NO
)AQNN was defined as 100%. See
``Experimental Procedures'' for
details.
Recognition Mechanism of the Substrates by MPP
In
the present study, we determined several critical residues or positions
in the extension peptide of malate dehydrogenase. We also found that
none of the peptides having five consecutive residues of the malate
dehydrogenase extension peptide sequence inhibited the processing
activity though they had the important sequences. This implies either
that the substrate-binding sites of the protease are multiple and
dependent each other or that the distal and proximal arginine residues
are spatially close by the aid of the proline and/or glycine and form a
specific structure in combination with the P
` amino acid to
present in the enzyme pocket the scissile bond to an active water on
the metal. We suppose that such structure should be a type of induced
fitting since the substrate peptides lack a secondary structure in
aqueous (without phospholipids or detergents) environment
as previously reported on other model extension
peptides(38) .
Implications for the Development of Real-time
Measurements of MPP Activity
Our recent establishment of the MPP
assay system that consists of synthetic peptides and HPLC enabled us to
study the recognition mechanism of MPP kinetically. Such a study has so
far virtually been impracticable in the conventional assay system with in vitro synthesized precursor proteins. The synthetic peptide
and HPLC system is, however, time-consuming and insensitive. Although
kinetic parameters obtainable with the new method described in this
paper are limited essentially to k
/K
, this substrate allows
an easy, rapid, sensitive, and accurate measurement of the reaction. The following observations demonstrate that cleavage of the
fluorogenic substrates by MPP is specific. (i) The cleavage point was
always Ser-Phe as shown in Fig. 3(Gly-Tyr for
P
`-replaced substrate, data not shown). (ii) The reaction
was not inhibited by a peptide corresponding to the mature potion of
malate dehydrogenase but strongly inhibited by peptides having partial
sequences of the extension peptide (Fig. 4). Especially, a
peptide with the proximal and distal arginine residues and flexible
linker was the most potent inhibitor. (iii) We can purify MPP from rat
mitochondria by only following the enzyme activity toward the
fluorogenic substrate throughout the purification process. Practically,
using the real-time measurement and employing preparative HPLC in
fractionation of the enzyme, we routinely obtain a highly pure form of
the enzyme in only 2 or 3 days.