Analysis of Elements in the Substrate Required for Processing by Mitochondrial Processing Peptidase*

We have recently demonstrated that synthetic pep- tides modeled on the extension peptide of malate dehydrogenase can be a good substrate of mitochondrial processing peptidase and that arginine residues present at positions (cid:50) 2 or (cid:50) 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 intramolecu-larly quenched fluorescent substrates. Using the fluoro- genic 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 1 (cid:42) -position. These results together with the previous data suggest that the proximal and distal arginine residues, proline and/or glycine between them, and P 1 (cid:42) amino acid could be critical determinants for the specific cleavage of the substrates by the protease. Most mitochondrial proteins synthesized on cytoplasmic ribosomes and into correct mitochondrial component. The majority of them carry N-terminal extension peptides that target the protein molecules to the organelle. (MPP) extension significant change of V max values (5.0 versus 8.4 pmol/min). These results indicate that the sequence from Ala 11 to Leu 13 was dispensable while at least one of the (cid:97) -helix-breaking amino acid residues (Pro 8 and Gly 10 ) was important.

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) 1 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 -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 -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 Ϫ2 or Ϫ3 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 1 Ј-position. 2

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 2 )) 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 -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 1 Ј were synthesized by a fragment condensation method. Briefly, a series of * This work was supported in part by Grant-in-aids for Scientific Research on Priority Areas No. 04259101 from the Ministry of Education, Science, and Culture of Japan. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. ‡ Present address: Dept. of Applied Chemistry, Nagasaki University, Nagasaki 852, Japan.
XSTY(NO 2 )AQNN peptides was synthesized on p-alkoxybenzyl-alcoholpolystyrene 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 1 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 2 )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-diphenylaminomethyl-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 fin ) after cleavage of the peptides with trypsin or chymotrypsin using ABz-Gly-NH 2 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 int ) of the substrates, were obtained. The k cat /K m values were determined from the 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 2 )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 l of 10% trifluoroacetic acid. After centrifugation at 10,000 ϫ g for 5 min, the supernatant was applied to a Cosmosil 5C18-AR column (10 ϫ 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 (Lä ufelfingen, Switzerland). Other reagents for peptide synthesis were from Watanabe Chemical Industries, Ltd. (Hiroshima, Japan). Fmoc-Tyr(NO 2 ) was prepared by coupling of Tyr(NO 2 ) (Janssen Chimica, Geel, Belgium) with Fmoc-Nsuccinimidyl 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. 1 shows 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 2 ) 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 ex-tension peptide, where Tyr(NO 2 ) was introduced at the 20th position (numbering from the original N terminus in premalate dehydrogenase), and ABz was connected to Met 1 . 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.
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 I). MPP cleaved the peptides that started from Ser 3 and Leu 5 as efficiently as that from Met 1 . It also cleaved the peptides that started from Arg 7 a little slower. The cleavage rates dropped dramatically for the peptides that lacked the sequences from Met 1 to Arg 7 . 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 2 ) acceptor at the 17th (P 1 Ј) or 18th (P 2 Ј) position. The peptide that had the acceptor at the P 1 Ј position (peptide 8 in Table I) had a drastically reduced cleavage efficiency compared to the peptide that had the acceptor at the 20th (peptide 1 in Table I). Introduction of Tyr(NO 2 ) into the P 2 Ј-position significantly elevated the cleavage efficiency when compared with the peptide having the acceptor at the P 1 Ј-position but not when compared with the peptide having the acceptor at the 20th position (compare peptides 9 and 3 in Table I). Since the substrates that connected ABz to Met 1 , Ser 3 , and Leu 5 were cleaved at nearly the same rate, we hereafter used ABz-LARPVGAALRRSFSTY(NO 2 )-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 1 , Leu 5 , and Arg 7 were separated by HPLC. Fig. 3 shows 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 2 ) 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 2 )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.
Deletion and Replacement of the Intervening Sequences between the Proximal and Distal Arginine Residues-The arginine residue that is located at the Ϫ2 or Ϫ3 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 II). Cleavage efficiencies of the substrates that lacked Ala 11 , Ala 11 -Ala 12 , and Ala 11 -Ala 12 -Leu 13 were essentially the same as ABzL5Y*20. The efficiency dramatically reduced, however, when deletion extended to Arg 14 . This seemed to be discrepant with our previous result that alanine was replaceable for Arg 14 (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 15 ) and distal (Arg 7 ) arginine residues. Replacement of Pro 8 with Gly did not significantly alter the cleavage efficiency of the peptide. However, replacement of Pro 8 and Gly 10 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 MLSALARAVAAAL-RRSFSTSA less efficiently than MLSALARPVGAALRRSF-STSA with K m values of 6.0 M (versus 0.7 M) without

TABLE II
Deletion and replacement of the intervening sequences between the proximal and distal arginine residues Cleavage efficiencies of the fluorogenic peptides that lacked the intervening sequences between the distal and proximal arginine residues and that had replacement of proline or proline and glycine were measured. See "Experimental Procedures" for details.

TABLE I Effects of the length of peptides and position of Tyr(NO 2 ) on self-quenching and cleavage efficiencies of the substrates
Derivatives of the extension peptide of rat malate dehydrogenase that had N-terminal deletions and Tyr(NO 2 ) at various positions were synthesized. ABz was attached to the N terminus. Tyr(NO 2 ) was introduced at P 4 Ј, P 1 Ј, or P 2 Ј. F int and F fin were fluorescence intensities measured for the substrates and for the final products obtained by hydrolysis with trypsin or chymotrypsin, respectively. The k cat /K m values were determined by the reaction with purified MPP using steady-state enzyme kinetics. See "Experimental Procedures" for details.

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 2 , PVGAA-NH 2 , AALRR-NH 2 , RRSFS-NH 2 , FSTSA-NH 2 , SAQNN-NH 2 ) that corresponded to the amino acid sequences from Leu 5 to Asn 24 . 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 2 ). We then synthesized longer peptides that elongated Nterminally from FSTSA-NH 2 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 M. Inhibition was enhanced when Arg-Arg-Ser was connected to FSTSA-NH 2 . Further elongation of RRSFSTSA-NH 2 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 2 at a position that corresponded to the distal position of the substrate, i.e. ARAAARRSFSTSA-NH 2 , 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 2 ). Among the peptides above, at least ARAAARRSFSTSA-NH 2 and ARPVGRRSFSTSA-NH 2 could act as substrates of MPP (data not shown).
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 impor-tance 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  II) supports importance of these amino acids.
Preference for Amino Acids at the P 1 Ј-position-The resistance to cleavage of the peptide having Tyr(NO 2 ) at P 1 Ј (Table I) suggested the limited acceptance of P 1 Ј residues in the substrate by MPP. To confirm this, we synthesized a series of peptides that had varied amino acids at the P 1 Ј-position. For synthetic convenience, the P 1 amino acid was replaced by Gly for Ser in the peptides. This conversion resulted in about half reduction of the cleavage efficiency. Fig. 5 shows the cleavage efficiencies of the peptides having altered P 1 Ј 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 1 Ј-position. Such amino acid residues are abundant at P 1 Ј 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 1 Ј for Neurospora MPP. They mutated phenylalanine at the P 1 Ј to lysine in a chimeric precursor of cytochrome b 2 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 1 Ј-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 1 Ј amino acid together with the proximal, distal, and flexible amino acids could determine the substrate specificity and cleavage position of MPP.
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 substratebinding 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 1 Ј amino acid to present in the enzyme pocket the scissile bond to an active Derivatives of ABzL5Y*20 that have Gly at P 1 (originally Pro) and various amino acid residues at P 1 were assayed. The activity toward ABz-LARPVGAALRRGFSTY(NO 2 )AQNN was defined as 100%. See "Experimental Procedures" for details.
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 3 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 cat /K m , 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 1 Ј-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.