©1995 by The American Society for Biochemistry and Molecular Biology, Inc.
Recognition of the Structure around the Site of Cleavage by the Carboxyl-terminal Processing Protease for D1 Precursor Protein of the Photosystem II Reaction Center (*)

Fumiko Taguchi (1), Yumiko Yamamoto (1), Kimiyuki Satoh (1) (2)(§)

From the (1) Department of Biology, Okayama University, Okayama 700, Japan and the (2) Division of Biological Regulation and Photobiology, National Institute for Basic Biology, Okazaki 444, Japan

ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
FOOTNOTES
REFERENCES

ABSTRACT

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, 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.


INTRODUCTION

The D1 protein is an integral component of the photosystem II (PSII)() 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.

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.


MATERIALS AND METHODS

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 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).


RESULTS

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.

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.


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 Kand 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 Kvalues 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 Kis 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.




DISCUSSION

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 -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.

The kinetic analysis of the reactions with COOH-terminal oligopeptides substituted at the +1 position indicated that rank order of V 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.

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 -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 Kvalue, although it greatly influences the V value. Thus, this position is probably not important for binding to the enzyme.

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.() 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 Kof 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



FOOTNOTES

*
This work was supported by a grant from the Mitsubishi Foundation; by Grants-in-aid for Scientific Research for Priority Areas (04273101), for Cooperative Research (04304004 and 05304006), and for General Research (06404003) from the Ministry of Education, Science and Culture of Japan; and also by a grant from the Human Frontier Science Program. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore by hereby marked `` advertisement'' in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

§
To whom correspondence should be addressed: Dept. of Biology, Faculty of Science, Okayama University, 3-1-1 Tsushima-naka, Okayama 700, Japan. Tel.: 81-86-251-7862; Fax: 81-86-255-3490.

The abbreviations used are: PSII, photosystem II; pD1, precursor to the D1 protein; HPLC, high performance liquid chromatography.

F. Taguchi, Y. Yamamoto, and K. Satoh, unpublished observation.


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