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J Biol Chem, Vol. 273, Issue 49, 32547-32553, December 4, 1998

Glutamate Residues Required for Substrate Binding and Cleavage Activity in Mitochondrial Processing Peptidase^*

Sakae Kitada, Katsuhiko Kojima, Kunitoshi Shimokata, Tadashi Ogishima, and Akio Ito^§

From the Department of Chemistry, Faculty of Science, Kyushu University, Fukuoka 812-8581, Japan

	ABSTRACT

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Mitochondrial processing peptidase, a metalloendopeptidase consisting of - and -subunits, specifically recognizes a large variety of mitochondrial precursor proteins and cleaves off N-terminal extension peptides. The enzyme requires the basic amino acid residues in the extension peptides for effective and specific cleavage. To elucidate the mechanism involved in the molecular recognition of substrate by the enzyme, several glutamates around the active site of the rat -subunit, which has a putative metal-binding motif, H⁵⁶XXEH⁶⁰, were mutated to alanines or aspartates, and effects on kinetic parameters, metal binding, and substrate binding of the enzyme were analyzed. None of mutant proteins analyzed was impaired in dimer formation with the -subunit. Mutation of glutamates at positions 79, 129, and 136, in addition to an active-site glutamate at position 59, resulted in a marked decrease in cleavage efficiency. Together with sequence alignment data, glutamate 136 appears to be involved in metal binding. Glutamate 129 is mostly responsible for the catalysis, as there was a considerable decrease in k_cat value by the mutation. Mutation of glutamate 79 led to decrease in k_cat value and increase in K_m values. Substrate binding experiments using an environmentally sensitive fluorescence probe attached to the peptide showed that the mutation caused a remarkable environmental change at the binding site to the N-terminal region of the substrate peptide and decreased binding of the peptide, thereby suggesting that glutamate 79 participates primarily in substrate binding. Thus, some glutamate residues required for substrate binding and cleavage activity have been identified.

	INTRODUCTION

Top Abstract Introduction Materials & Methods Results Discussion References

Most nuclear-encoded mitochondrial proteins are synthesized on cytoplasmic ribosomes as larger precursors with N-terminal extension peptides for targeting into mitochondria (1-3). During or after import of the precursors into mitochondria, the extension peptides are proteolytically removed by three types of processing peptidases. Mitochondrial processing peptidase (MPP¹; EC 3.4.24.64) (4-8) generally cleaves off a large part of the extension peptide, including the mitochondrial matrix-targeting sequence as the initial step of the processing. Many precursors are converted into the mature forms by the one-step processing. The second enzyme is mitochondrial intermediate peptidase, which catalyzes second-step cleavage in the two-step processing of some precursor proteins (9, 10). These precursors are first cleaved by MPP, and then the residual octapeptides are removed by the mitochondrial intermediate peptidase. The last enzyme is inner membrane protease I, which processes the signal sequence for inner membrane and intermembrane space (11). The last two peptidases sequentially act after cleavage of the matrix targeting sequences by MPP. Thus, MPP plays an important role in proteolytical processing of precursors in mitochondria.

MPP has been purified from mitochondria of Neurospora crassa (12), yeast Saccharomyces cerevisiae (13), rat liver (14), and a few plants (15, 16). The enzymes, except for those of the plant, are soluble proteins in the mitochondrial matrix and consist of  and  subunits. The subunits from yeast and rat liver form a stable heterodimer, whereas Neurospora subunits could be separated from each other by gel filtration. Processing activity of the enzymes is sensitive to metal chelators, and the lost activity is restored by divalent metal ions (4-7). MPP specifically recognizes a large variety of mitochondrial precursor proteins and cleaves off the extension peptides at single sites (14, 17, 18). Contrary to the strict substrate specificity of the enzyme, amino acid sequences of the extension peptides are wide in length and poor in similarity (1). Many experimental observations have indicated that basic amino acid residues in the extension peptides were required for the effective processing by MPP (17-20). A consensus sequence of the processing signals has remained to be established. MPP may well recognize some higher order structure of extension peptide with the basic amino acids.

When primary structures of two subunits of MPP from yeast, Neurospora, rat, and potato were determined (12, 15, 21-28), each subunit proved to be highly homologous among organisms. The subunits of MPP also have significant sequence homology with a family of endopeptidases, the pitrilysin family (29, 30), that includes Escherichia coli pitrilysin (also called protease III), the insulin-degrading enzymes from mammals and insects, the N-arginine dibasic convertase from rat. Despite metalloendopeptidases, they lack the thermolysin-like zinc binding motif HEXXH, but all the members except the -MPPs have the inverted motif, HXXEH. Site-directed mutagenesis showed a zinc binding site in E. coli pitrilysin (31). The - and -subunits of the MPP are also homologous to core proteins, core 2 and core 1, respectively, of mitochondrial ubiquinol-cytochrome c oxidoreductase (bc₁ complex), a component of the respiratory chain (32). In potato and spinach mitochondria, interestingly, two subunits are identical with the core proteins and are integrated into the bc₁ complex (15, 16).

Attempts to obtain each subunit by expression in E. coli demonstrated that each subunit alone had no activity. Thus, cooperative actions of the two subunits are essential for the processing activity (33-35). Mutation experiments at the putative metal binding site, His^56-Xaa-Xaa-Glu⁵⁹-His⁶⁰ in the -MPP, demonstrated that this subunit is a catalytic subunit (36). In addition, amino acid residues and regions in the -MPP seem to be functionally important, as deduced from mutation studies (37, 38).

We have now identified amino acid residues involved in the enzyme function, and we attempted to elucidate mechanisms involved in molecular recognition of substrates by the enzyme. Several glutamates around the active site of the rat -subunit were mutated to alanines or aspartates, and effects on kinetic parameters, metal binding, and substrate binding of the mutated enzymes were analyzed. We find that some glutamate residues are indeed essential for substrate binding and catalytic reaction.

	MATERIALS AND METHODS

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Construction of Expression Vectors and Site-directed Mutagenesis of MPP-- pTrc, which contains -, and -MPP cDNAs in tandem, was constructed by insertion of cDNAs from pET-3a- (36) into a multiple cloning site of into pTrc99A plasmid (Amersham Pharmacia Biotech). Pre-adrenodoxin (preAd) cDNA in pET3d plasmid, pET3dPAD, was inserted into the tetracycline-resistant region of pACYC184 (Nippon Gene) to obtain pACETPAD.

To introduce mutations at glutamate residues (E47, E75, E77, E79, E129, E136, and E139) in -MPP, site-directed mutagenesis was done using the Kunkel method (39). The mutated cDNAs were ligated into pTrc99A, as described above.

Co-expression of MPP and preAd in E. coli-- BL21(DE3) cells transformed with pTrc and pACETPAD were selected with both chloramphenicol and ampicillin, and the resultant cells were cultured in 0.5 mM isopropyl--D-thiogalactopyranoside for 20 h at 25 °C. After harvesting the cells by centrifugation for 10 min at 1,000 × g, the cells were solubilized in SDS sample buffer, and the solubilized cell extract was applied to SDS-polyacrylamide gel electrophoresis followed by Western blotting using anti--MPP, anti--MPP, and anti-Ad antibodies. In vivo processing of preAd by MPP was estimated from conversion of preAd to the intermediate form.

Purification and Characterization of Histidine-tagged MPP Subunits-- A hexahistidine tag was introduced into the C termini of rat -MPP and yeast -MPP using polymerase chain reaction. The amplified cDNAs for histidine-tagged subunits were inserted into pTrc99A, and the proteins were expressed as described above. After sonication of the cells, the soluble extract was recovered by centrifugation for 20 min at 15,000 × g. The extract was loaded onto a nickel-chelating Sepharose column (Amersham Pharmacia Biotech; His trap) equilibrated with 10 mM HEPES-KOH, pH 7.5, containing 500 mM NaCl, 10 mM imidazole, 30% glycerol, and 0.01% Tween 20. After washing the column with the buffer, histidine-tagged MPP subunits were eluted with 500 mM imidazole. Some mutant -MPPs were further purified using a DEAE Toyopearl column. The purity was confirmed by SDS-polyacrylamide gel electrophoresis.

In Vitro Assays of MPP Activity-- Kinetic parameters were determined using a synthetic peptide, MDH 1-21, a model based on extension peptide of mouse malate dehydrogenase (MDH) as a substrate (17). The purified wild-type or mutant -MPP was mixed with purified yeast -MPP, then the in vitro reconstituted MPPs were incubated with the synthetic peptide in 400 µl of 20 mM HEPES-KOH buffer, pH 7.4, containing 0.1% Tween 20 at 25 °C. The reaction was stopped by adding 0.05% trifluoroacetic acid. The processing products were analyzed by reversed phase high performance liquid chromatography, as described (17). K_m and k_cat values for MPPs were determined from Lineweaver-Burk plots. Enzyme activity was also determined by continuous fluorometric assay described by Ogishima et al. (40), using amino benzoyl-LARPVGAALRRSFSTY(NO₂)AQNN peptide as substrate. The recombinant MPPs were incubated with the fluorogenic peptide in 20 mM HEPES-KOH buffer, pH 7.4, containing 0.01% Tween 20 at 25 °C. The reaction was monitored at 420 nm (excitation at 315 nm).

Metal Determination-- We quantified metal ion in each protein using inductively coupled plasma mass spectrometer (Yokogawa, model PMS 2000). To detect metal ions specifically bound to proteins, the purified proteins were dialyzed against 10 mM HEPES-KOH, pH 7.4, containing 50 mM NaCl and 0.01% Tween 20. After dialysis, the solution containing 10 to 100 µg of the protein was diluted with 0.1 N HNO₃ and subjected to determination metals.

Binding of Fluorescence-labeled Peptide to MPP-- The MDH 5-25 peptide was labeled at the N-terminal -amino group with 7-diethylaminocoumarin (DAC), N-DAC-MDH. The MDH 5-25 (24K) peptide that had been acetylated at the -amino group was also introduced by DAC into the -amino group of lysine residue at position 24 to obtain C-DAC-MDH. The coumarin-labeled peptides of indicated concentrations were added to the purified MPP and preincubated with 5 mM EDTA on ice for 30 min. The fluorescence change was monitored at 470 nm (excitation at 390 nm) at 25 °C using a Hitachi F-2000 fluorescence spectrophotometer. K_d values were calculated as follows, F = (F_max [L])/(K_d + [L]), where F and F_max are the measured and maximal fluorescence intensity of the peptides, respectively, and [L] is the total peptide concentration. A plot of [L]/F versus [L] yields a linear function with a slope of 1/F_max and an ordinate intercept of K_d.

	RESULTS

Top Abstract Introduction Materials & Methods Results Discussion References

Site-directed Mutation of Glutamate Residues in the N-terminal Portion of -MPP-- To identify residues responsible for substrate recognition and catalytic reaction, we focused on glutamic acids in the N-terminal region of -MPP, because this region contains a putative active-site sequence, HXXEH, and has a significant degree of homology to members of the pitrilysin family (Fig. 1). There are two kinds of glutamates in the region, well conservative residues among peptidases of this family and residues characteristic of -MPP. Glutamates corresponding to residues at positions 129 and 136 of rat MPP are completely conserved. A glutamate at position 47 is also conserved as an acidic residue. On the other hand, a segment of 7 amino acids from position 74 to 80, Leu-Glu⁷⁵-Leu-Glu⁷⁷-Ile-Glu⁷⁹-Asn, is characteristic of -MPP and is a variable region for other peptidases. This segment has a characteristic structure in which three glutamic acids are positioned in hydrophobic sequence of leucine and isoleucine residues at every other residue. Glutamate at position 139 is also characteristic of -MPP. We then focused on glutamate residues at positions 47, 75, 77, 79, 129, 136, and 139 and individually mutated them to alanines or aspartates to determine the role of these residues in substrate recognition and/or catalytic reaction.

View larger version (65K): [in this window] [in a new window]   Fig. 1.   Alignment of N-terminal regions of -MPPs and several members in a new superfamily of metalloendopeptidase (pitrilysin family). Numbers to the left of the sequences of -MPPs indicate positions in mature proteins. Numbers of the other sequences show positions in proteins deduced from the open reading frames of the corresponding nucleotide sequences. At least two identical residues among the members are shaded. The putative metal binding sites, HXXEH, are printed in white on black. Numbers on the alignment indicate positions mutated in this study. Dashed areas indicate gaps to maximize identity. Multiple alignment of sequences was performed using Clustal W of the computer program. N.c., N. crassa; PTR, pitrilysin; IDE, insulin-degrading enzyme; NAD convertase, N-arginine dibasic convertase.

In Vivo Processing of Precursor Protein by Wild-type and Mutant MPPs in E. coli-- For a simple and rapid detection of processing activity of recombinant MPP, we used a multi-expression system, in which two MPP subunits and a precursor protein, preAd, were co-expressed in E. coli cells, and in vivo processing was analyzed by measuring conversion of the precursor to the intermediate protein, which is a form of adrenodoxin processed by MPP. When both subunits of the enzyme were co-expressed, a single band of the intermediate form was observed in the cell, thereby indicating complete processing of the co-expressed precursor to the processed form (Fig. 2A). Cells expressing - or -MPP alone as the enzyme protein exhibited no processed protein band, even though they had bands between the precursor and processed bands, which may have been the products nonspecifically cleaved by endogenous bacterial proteases because those bands were observed even in the cell without MPP cDNAs.

View larger version (70K): [in this window] [in a new window]   Fig. 2.   Processing of pre-adrenodoxin by the wild-type (WT) and mutant MPPs in E. coli. A, detection of processing activity of wild-type MPP using co-expression of preAd and MPP. The transformation, cell growth, and Western blotting were done as described under "Materials and Methods." Intermediate Ad (iAd) expressed in E. coli was used as a molecular weight marker to estimate the preAd processed by co-expressing MPP in E. coli. Control plasmid means a plasmid pTrc without any inserted gene. The asterisk indicates nonspecific degradation products of preAd by endogenous bacterial peptidase(s). Correct conversion of pre-Ad to the intermediate form of Ad (iAd) was confirmed by N-terminal sequencing of the processed product. B, processing activity of MPPs mutated at highly conserved amino acid residues among the pitrilysin family. C, processing activity of MPPs mutated at three glutamates in the negatively charged segment characteristics of rat -MPP. The co-expression assay was done at least three times for all of the mutants, and the processing efficiency was denoted based on the average. Activity levels; +++, >50% conversion of the precursor to the intermediate form; ++, 25-50% conversion; +, 5-25% conversion; , no conversion.

Using this system, we analyzed efficiency of in vivo processing of preAd in the cells expressing mutant MPPs (Fig. 2, B and C). As expected from earlier results (36), mutation of the amino acid residues forming the putative active site, His-56, Glu-59, and His-60, resulted in complete loss of processing. Cells expressing mutants at positions 79 and 136 also exhibited no processing activity, even when glutamates were changed to aspartates. These glutamates as well as two histidines involved in metal binding seem to be essential for the processing. No detectable band of the processed protein was observed in cells expressing mutants of glutamates at position 47 and 129 to alanines, whereas cells expressing the mutants to aspartates had slight processing activity, suggesting that these glutamates function as acidic amino acids. For other glutamates at positions 75, 77, and 139, mutation to either alanine or aspartate had little effect on processing reactions.

In Vitro Reconstitution of MPP-- For quantitative analysis of functions of these glutamates, we wanted to purify recombinant rat MPP from E. coli cells. However, most rat -MPP was produced as inclusion bodies or insoluble aggregates in the cell, in contrast to the -MPP subunit that was in mostly soluble form, and the amount of the active dimer was insufficient for purification. We then used yeast -MPP, most of which was expressed in a soluble monomeric form, as the partner to the mutant rat -MPP.

Because the addition of the hexahistidine tag at the C-terminal end of either subunit had no apparent effect on processing activity of the wild-type enzyme, we introduced the tag to both subunits and individually expressed them in E. coli cells. After purification of each subunit so as to be homogeneous with Ni²⁺-chelating column, two subunits were mixed to reconstitute the active enzyme. As rat wild-type -subunit was added to yeast -subunit, the processing activity was increased, and the maximum activity was obtained at approximately equal moles of the subunits (data not shown). The reconstituted enzyme exhibited processing activity to various precursor proteins, including aspartate aminotransferase, cytochrome P-450(SCC), and malate dehydrogenase, translated in vitro, but each subunit alone had no activity (data not shown). When kinetic parameters for the reconstituted enzyme were determined using a synthetic oligopeptide based on the N-terminal sequence of precursor of MDH, MDH1-21, as substrate, the k_cat and K_m values were calculated to be 84 min¹ and 74 nM, respectively, and the values were essentially the same as those for the purified bovine liver MPP. Thus, the heterogenously reconstituted enzyme seems to have much the same nature as the native enzyme obtained from mammalian mitochondria.

Effect of Mutation of -MPP on Association to -MPP-- To determine whether loss of processing activity of the mutant enzymes shown in Fig. 2, B and C would result in failure in association of the subunits, we examined the association of the wild-type and mutated  subunits to the histidine-tagged  subunit on the nickel-chelating column (Fig. 3). After a bacterial extract expressing rat -MPP was mixed with the purified histidine-tagged yeast -MPP, the mixture was applied onto a nickel-chelating column, and the adsorbed proteins were eluted with 0.5 M imidazole. The wild-type -MPP bound to the column and co-eluted with the histidine-tagged -MPP, whereas it did not elute in the absence of tagged -MPP. The figure shows a nearly 1 to 1 ratio of subunits in the MPP. Moreover, no increase in the activity was observed with the addition of the  or  subunit to the eluted enzyme preparation, and molecular mass of the eluted enzyme was estimated by gel filtration to be approximately 100 kDa (data not shown). Thus, two subunits eluted from the column were fully assembled as the heterodimer. When the mutated  subunits were subjected to the same procedure, all the mutant proteins co-eluted with the histidine-tagged  subunit. The subunit ratios were similar to that of the wild-type enzyme, and the molecular size of the complex was also about 100 kDa (data not shown). Thus, all the mutant  subunits examined here could associate with the  protein to form a heterodimer.

View larger version (70K): [in this window] [in a new window]   Fig. 3.   Association of yeast -MPP and rat -MPP. We detected interaction between the - and -MPPs using a Ni2+-chelating column. Bacterial extract containing rat -MPP ( extract) was mixed with purified yeast hexahistidine-tagged -MPP ( His × 6), then the mixture was applied onto a Ni2+-chelating Sepharose column. After washing the column with HEPES-KOH buffer, pH 7.5, proteins were eluted with the buffer containing 500 mM imidazole (left panel). The extract containing rat -MPP alone was also subjected to the same procedure (right panel). Eluted proteins were analyzed by SDS-polyacrylamide gel electrophoresis followed by staining with Coomassie Brilliant Blue R-250. Bacterial extract containing the mutated -MPP and the histidine-tagged -MPP were also mixed and then applied onto a Ni2+-chelating Sepharose column to detect the subunits association.

We have shown that the addition of the inactive  subunit mutated at the putative active site efficiently decreased the wild-type MPP activity by replacing the wild-type  subunit (36). We then applied this system for quantitative analysis of the interaction between the wild-type  subunit and the mutant  subunits, E79A and E136A, whose activities were not detected in in vivo processing (Fig. 2B). As shown in Fig. 4, the mutant subunits clearly had a decreased MPP activity, and 50% reduction was observed at equimolar wild-type and mutant  subunits. Thus, the mutant  subunits can bind to the  subunit with a similar affinity to the wild type. The results indicate that loss of the processing activity observed in some mutants is not because of failure of formation of a stable heterodimeric complex.

View larger version (23K): [in this window] [in a new window]   Fig. 4.   Inactivation of MPP by addition of mutated  subunits. Purified wild-type (5 ng) and mutant (from 5 to 50 ng)  subunits were first mixed, then the purified  subunit (5 ng) was added to the mixture. The activity of the wild-type MPP complex was measured using a fluorogenic peptide, Aminobenzoyl-MDH, as substrate at 25 °C. The remaining activity is given as the percentage of activity in the absence of the added (add) wild-type (wt)  (filled squares), H60R (filled circles), E79A (open squares), or E136A (open circles) subunit.

Determination of Metal in Wild-type and Mutant -MPP Subunits-- To determine whether these histidine and glutamate residues conserved through the pitrilysin family are directly involved in metal binding, we measured the metal ion in wild-type and some mutant  subunits using inductively coupled plasma mass spectrometer. Because qualitative analysis using the mass spectrometer showed that only Ni²⁺ was detected in significant amounts in the wild-type  subunit purified with a Ni²⁺-chelating column instead of Zn²⁺ (which was detected in the protein purified using conventional nonchelating columns), Ni²⁺ must have replaced with Zn²⁺ during purification. This result means that replacement of metal has no effect on the catalytic activity of the enzyme. The molar ratios of Ni²⁺ per protein are shown in Table I. Because the  subunit has no putative metal binding sequence, the amount of the metal observed in this subunit must be background in the present assay system. Only background level of Ni²⁺ was detected in the two histidine mutants, His-56 and His-60, whereas two glutamate mutants, Glu-59 and Glu-79, were demonstrated to bind nearly stoichiometric amounts of the metal, as in the case of the wild-type enzyme. On the other hand, mutation at Glu-129 and Glu-136 constantly decreased the amount of the metal bound to the enzyme, although to not a large extent, suggesting participation of these glutamates in metal binding. Because the Glu-136 mutant was more affected by mutation both in the catalytic activity and metal binding than the Glu-129 one, it is likely that Glu-136 participates in the metal coordination in the enzyme.

Effect of Mutation of Glutamate Residues on Kinetic Parameters-- To examine the effects of mutations on the substrate recognition and catalytic activity, we determined kinetic parameters of the mutant enzymes, using the synthetic peptide substrate, MDH1-21. Table II shows effects of mutation of glutamate residues conserved among peptidases in the pitrilysin family, on k_cat and K_m values. No activity was detected in the enzymes mutated at His-56, Glu-59, His-60, and Glu-136, even when a large amount of the enzymes or higher concentrations of the substrate were used. Mutation of glutamate at position 47 to aspartate had practically no effect on the K_m value and only slightly affected the k_cat value, whereas mutation to alanine resulted in a decrease in the k_cat value and an increase in K_m value so that the catalytic efficiency, k_cat/K_m, was dramatically reduced to about 5% that of of the wild-type enzyme. Replaceability of glutamate with aspartate at this position seems to be reasonable, because the aspartate is conserved among other related peptidases (Fig. 1). Thus, acidic amino acids at this position may function in these peptidases, probably both in catalytic reaction and substrate binding. Mutations at Glu-129 had little effect on K_m values, yet resulted in a 10-20-fold decrease in k_cat values. The residue seems to be responsible for catalysis rather than for the substrate binding. An alanine mutation at Glu-139 located near the conserved Glu-129 and -136 had no effect on both kinetic parameters, indicating that the residue is not essential for MPP activity.

View this table: [in this window] [in a new window]   Table II Effect of substitutions at conserved glutamates in -MPP The kinetic parameters of the wild-type and mutant MPPs were measured using MDH1-21 peptide as substrate, as described under "Materials and Methods" and calculated from Lineweaver-Burk plots.

Because our previous studies on structural features common to the substrates of MPP revealed that basic amino acids are essential for recognition of the substrates by the enzyme (17-20), an acidic amino acid cluster that is characteristic of the MPP among pitrilysin families, Glu-75, Glu-77, and Glu-79, is a possible candidate as a segment responsible for substrate recognition. Table III shows the effect of mutation of glutamates on kinetic parameters. Although we could not detect processing activity for the E79A and E79D mutants in an in vivo assay, extremely low but definite activity was detected in the in vitro assay. Mutation of the glutamate resulted in a considerable decrease in both catalytic activity and the affinity to substrate, indicating that Glu-79 plays an essential role characteristic of the MPP. The mutations at other glutamates in the acidic amino acid cluster caused only a slight increase in K_m value.

Requirement of the Glutamate 79 for Binding to Extension Peptide-- To determine the effects of the mutation on direct interactions between the enzyme and substrate, we synthesized the fluorescence-labeled peptides, in which an environment-sensitive coumarin derivative was covalently introduced into the peptide (MDH1-21) at the N- or C terminus (N-DAC-MDH or C-DAC-MDH, respectively), and we directly analyzed the substrate binding ability of the enzymes. The coumarin-labeled peptide gave a fluorescence emission spectrum with the maximum at 482 nm, and the addition of the MPP, which had been inactivated in the presence of EDTA, led to a large increase in fluorescence intensity and a blue shift in emission spectrum (emission maximum at 470 nm) (data not shown). Titration of the wild-type MPP with N-DAC-MDH and C-DAC-MDH exhibited a simple saturation curve of emission intensity at 470 nm (Fig. 5, filled squares). The figure also shows that an extremely small increase in fluorescence emission with N-DAC-MDH was detected in Glu-79 mutants, E79A (open circles) and E79D (filled circles), under the same condition as for the wild-type enzyme. On the other hand, only a small difference in the maximum level of the fluorescence intensities was observed between wild-type and mutant enzymes with C-DAC-MDH. This means that the environment around the binding site of the N-terminally attached fluorophore was probably greatly changed by the mutation.

View larger version (15K): [in this window] [in a new window]   Fig. 5.   Binding of DAC-labeled MDH peptides to MPP. Fluorescence emission increases (F = Fobs  Fo) of N-DAC-MDH (panel A) or C-DAC-MDH (panel B) peptide by wild type (filled squares), E79A (open circles), or E79D (filled circles) mutant MPPs are measured at increasing concentrations of peptides. F was calculated from fluorescence intensities measured in the absence (Fo) and presence (Fobs) of the enzyme at each concentration of peptides. 1.0 µM of enzymes that had been treated with 0.5 mM EDTA to remove the active center metal was used.

The same titration experiment was carried out for other mutant enzymes, and the dissociation constants, K_d, were calculated from simple saturation curves of emission intensity at 470 nm, as described under "Materials and Methods." Table IV shows that wild-type MPP had practically the same affinity to N-DAC-MDH and C-DAC-MDH peptides, 48 and 46 nM, respectively, and the values were also similar to the K_m value, 74 nM, determined above. Substantially the same K_d values of N-DAC-MDH and C-DAC-MDH peptides were obtained with the Glu-79 mutants, and the values were about 20-fold higher than that for the wild-type. The other mutant enzymes analyzed exhibited similar K_d values for N-DAC-MDH peptide to the wild-type. These results indicate that glutamate 79 seems to be involved in interaction with the substrate, particularly of the N-terminal portion of the extension peptide.

	DISCUSSION

Top Abstract Introduction Materials & Methods Results Discussion References

We substituted seven glutamate residues in the N-terminal region of rat -MPP in an attempt to identify residues involved in the enzyme function. Kinetic and subunit interaction analysis revealed that Glu-79, Glu-129, and Glu-136 were responsible for the enzymatic activity but not for subunit assembly.

We have shown that two histidines and a glutamate in the HXXEH motif in the  subunit are essential for MPP activity, probably as a metal binding and catalytic site (36). Here we clearly demonstrated direct and no participation of the two histidines and glutamate, respectively, in metal binding. The finding agrees with those in pitrilysin (31). Thus, the function of HXXEH motif is highly conserved as the metal binding through the pitrilysin family. Based on sequence homology with other members of pitrilysin family, Glu-136 may be a candidate of the third metal binding site in the  subunit. Site-directed mutation experiments at Glu-136 clearly demonstrated that the mutation to either aspartate or alanine led to a complete loss of the activity. Alanine mutant at the Glu-136, however, contained low but significant amounts of the metal compared with the histidine mutants described above. Similar results were reported in the pitrilysin. Mutation at the Glu-169 in pitrilysin, which is a corresponding residue to Glu-136 in MPP and is proposed to the third metal binding site, also resulted in loss of the insulin-degrading activity but still retention of significant amount of the metal (41). Thus, the proposed third metal binding site may have lower responsibility for metal coordination than the histidines in HXXEH motif. Physiochemical and structural analysis will be needed to elucidate the metal coordination state in the members of pitrilysin family.

Because Glu-129 is completely conserved in the pitrilysin family, it may play another role in the catalysis of metalloendopeptidases, such as stabilization of the tetrahedral intermediate in the proteolytic reaction. The mutation at the position in pitrilysin and insulin-degrading enzyme greatly reduced the total activities (41, 42). The present kinetic analyses demonstrated that the glutamate seemed to be required for effective turnover but not for substrate binding. We cannot, however, completely exclude the possibility that the mutations affected conformation of the -MPP. Indeed, mutation of the Glu-129 tended to decrease the protein stability in E. coli. Structure prediction of the protein tells us that mutation of the glutamate to alanine or aspartate leads to disruption of the -helical structure around the segment that contains a putative metal binding residue, Glu-136. Thus, the mutation could influence the metal binding conformation, and consequently, the k_cat value of the mutant protein might decrease, without affecting subunit assembly and substrate binding.

An acidic amino acid cluster characteristic of MPP among pitrilysin families, Glu-75, Glu-77, and Glu-79, is a possible candidate as a segment responsible for substrate recognition, because previous studies on structural features common to the substrates of MPP revealed that basic amino acids are essential for recognition of the substrates by the enzyme (16-20). Mutation of the glutamates demonstrated that Glu-79 is a key residue for effective cleavage of the substrates and that Glu-75 and Glu-77 do not seem to be essential for the activity. Binding affinity of the peptide to Glu-79 mutant was reduced to a similar extent between N- and C-terminal-labeled peptides. Interestingly, the fluorescence emission from an environment-sensitive probe attached to the N-terminal portion induced by interaction with the mutant MPP was markedly decreased, although little effect was observed on the emission with the C-terminal-labeled peptide. These findings suggest that mutation at Glu-79 causes a remarkable environmental change in the binding site for the N-terminal region of the substrate peptide, and that failure in correct interaction of the region of the peptide to MPP brings about impairment of leading and fixing the cleavage site of the peptide to the correct position in the active site of MPP; consequently, the rate of the cleavage reaction would be markedly decreased.

The - and -subunits of the MPP are homologous to the core 2 and core 1 proteins of mitochondrial ubiquinol-cytochrome c oxidoreductase (bc₁ complex), a component of the respiratory chain (30). the Crystal structure of the bc₁ complex from bovine heart mitochondria has recently been reported (43), and one can expect that MPP is similar in structure to the complex. Although detailed data are not available, core 1 and core 2 proteins are structurally similar and consist of two domains of roughly equal size and almost identical folding topology. In dimer form, they form a crack leading to the internal cavity, the wall of which is lined with mostly hydrophilic amino acid residues. Considering that arginine residues in extension peptides are required for substrate recognition by the MPP (17-20), the substrate binding pocket of the MPP is likely to be rich in negatively charged amino acids. Thus, the functional glutamates and metal binding sites of the -subunit are probably arranged in the crack or cavity.

Because MPP recognizes several structural features in precursor proteins, including basic amino acid residues proximal and distal to the cleavage site and hydrophobic residue at position 1 (17-20, 40), the enzyme is likely to have multiple binding sites corresponding to these structural elements. It is most likely that the amino acids around the cleavage site interact with the -subunit, because this subunit has the active-site metal and catalytic residue, Glu-59. On the other hand, Glu-79 seems to function either to interact directly with or to lead the basic amino acid residues in the extension peptide to the proper position of the -subunit. Basic amino acid residues at the upper N-terminal region of the extension peptide may interact with the -subunit since our recent results show that mutation in the -subunit leads to a decrease in cleavage efficiency of precursors with a longer extension peptide (44). In the model, the flexible linker regions containing proline and glycine in the portion between proximal and distal arginines (40, 45) seem to help fit the substrates to the binding sites of the MPP subunits. Thus, cooperative interactions at multiple sites strengthen substrate recognition and make it specific even if interaction at each site is not so strong in the binding of the substrates. The multiple-site recognition mechanism may make feasible strict specificity and high affinity to MPP for precursors, the structures of which have little in common.

	ACKNOWLEDGEMENTS

We are greatly indebted to Dr. Midori Watanabe, the Center of Advanced Instrumental Analysis, Kyushu University, for determination of metal ion using inductively coupled plasma mass spectrometer and to Dr. Shun-ichiro Kawabata for N-terminal sequencing of Ad protein processed by MPP. We also thank Dr. Masa-aki Ohba for discussion about metal detection.

	FOOTNOTES

^* This work was supported in part by grants-in-aid for Scientific Research from the Ministry of Education, Science, Sports, and Culture of Japan (to A. I., T. O., and S. K.) and for Core Research for Evolutional Science and Technology in Japan (to A. I.).The costs of publication of this article were defrayed in part by the payment of page charges. The 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 Biochemistry, School of Medicine, Keio University, Tokyo 160-8582, Japan.

^§ To whom correspondence should be addressed. Tel. and Fax: 81-92-642-2530; E-mail: a.itoscc{at}mbox.nc.kyushu-u.ac.jp.

The abbreviations used are: MPP, mitochondrial processing peptidase; -MPP, subunit of MPP; -MPP, subunit of MPP; Ad, adrenodoxin; preAd, precursor of Ad; DAC, 7-diethylaminocoumarin-3-carbonic acid; MDH, malate dehydrogenase.

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