Tài liệu Báo cáo khoa học: Kinetic basis for linking the first two enzymes of chlorophyll biosynthesis doc

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Tài liệu Báo cáo khoa học: Kinetic basis for linking the first two enzymes of chlorophyll biosynthesis doc

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Kinetic basis for linking the first two enzymes of chlorophyll biosynthesis Mark Shepherd, Samantha McLean and C. Neil Hunter Robert Hill Institute for Photosynthesis and Krebs Institute for Biomolecular Research, Department of Molecular Biology and Biotechnology, University of Sheffield, UK Magnesium chelatase lies at a branch point in tetrapyr- role biosynthesis where insertion of Mg 2+ eventually results in the production of chlorophyll, or the insertion of Fe 2+ produces heme. Magnesium chelatase is com- prised of three protein subunits, ChlI (38–42 kDa), ChlD (60–74 kDa) and ChlH (gun5, 140–150 kDa) (BchIDH in photosynthetic bacteria) [1–4]. ChlI is an AAA + ATPase [5,6], contains a Mg 2+ binding site [7], and forms a stable complex with ChlD [8]. The third subunit, ChlH, binds porphyrins [9,10] and presumably contains the active site for chelation. The steady-state kinetic characterization of magnesium chelatase quanti- fied the ATP hydrolysis required to complete a catalytic cycle and revealed a cooperativity with respect to Mg 2+ , which has important implications for regulation of chlo- rophyll biosynthesis [11]. The structure of Gun4, a pro- tein that binds to the tetrapyrrole substrate and product of the magnesium chelatase, has recently been solved [12]. Kinetic analysis revealed that Gun4 dramatically enhances the magnesium chelatase reaction, and reduces the threshold Mg 2+ concentration required for chelatase activity at low substrate concentrations, implying a possible role for this protein in substrate delivery. The next step in chlorophyll biosynthesis, catalysed by magnesium protoporphyrin IX methyltransferase (ChlM in Synechocystis), involves the transfer of a methyl group from S-adenosyl-l-methionine (SAM) to the propionate group on ring C of magnesium proto- porphyrin IX (MgP) to form magnesium protopor- phyrin IX monomethylester (MgPME). Steady-state kinetic assays showed that the reaction proceeds via a random binding mechanism forming a ternary complex [13]. Stopped-flow fluorescence studies indicated that a relatively slow ( 70 s )1 ) domain reorganization of ChlM alters the conformation of the MgD binding site and precedes rapid (> 600 s )1 ) substrate binding (K d 3.36 lm) [14]. Rapid quenched-flow analysis showed that a catalytic intermediate is formed and Keywords chlorophyll; chelatase; methyltransferase; gun signalling Correspondence M. Shepherd, Department of Biochemistry and Molecular Biology, A222 Life Sciences Building, Green Street, University of Georgia, Athens, GA 30602, USA Fax: +1 706 5427567 Tel: +1 706 5427252 E-mail: shepherd@secsg.uga.edu (Received 25 February 2005, revised 5 July 2005, accepted 19 July 2005) doi:10.1111/j.1742-4658.2005.04873.x Purified recombinant proteins from Synechocystis PCC6803 were used to show that the magnesium chelatase ChlH subunit stimulates magnesium protoporphyrin methyltransferase (ChlM) activity. Steady-state kinetics demonstrate that ChlH does not significantly alter the K m for the tetrapyr- role substrate. However, quenched-flow analysis reveals that ChlH dramat- ically accelerates the formation and breakdown of an intermediate in the catalytic cycle of ChlM. In light of the profound effect that ChlH has on the methyltransferase catalytic intermediate, the pre steady-state analysis in the current study suggests that ChlH is directly involved in the reaction chemistry. The kinetic coupling between the chelatase and methyltrans- ferase has important implications for regulation of chlorophyll biosynthesis and for the availability of magnesium protoporphyrin for plastid-to-nucleus signalling. Abbreviations Mg chelatase, magnesium chelatase; MgD, magnesium deuteroporphyrin IX; MgDME, Mg deuteroporphyrin IX monomethyl ester; MgP, magnesium protoporphyrin IX; MgPME, Mg protoporphyrin IX monomethyl ester; Mops, 4-morpholinepropanesulfonic acid; P IX , protoporphyrin IX; SAH, S-adenosyl- L-homocysteine; SAM, S-adenosyl-L-methionine; Synechocystis, Synechocystis PCC6803. 4532 FEBS Journal 272 (2005) 4532–4539 ª 2005 FEBS then depleted (rate constants of 11.9 ± 0.5 s )1 and 11.8 ± 0.5 s )1 , respectively), and the decay of the intermediate species coincides with the evolution of magnesium deuteroporphyrin monomethylester (MgDME) product, which implies that MgDME is formed via the decay of this species [14]. The tetrapyrrole substrate (MgP) and product (MgPME) for the ChlM catalysed reaction have been implicated in plastid-to-nucleus signalling [15–20]. As MgP is also the product of the magnesium chelatase reaction, this emphasizes the importance of quantita- tive studies not only of the methyltransferase and chelatase, but also of the interaction between these enzymes. The coupling of the magnesium chelatase and MgP methyltransferase steps is not a new idea; in 1962, inhibition of methyltransferase activity by ethio- nine resulted in the accumulation of coproporphyrin (rather than MgP) by whole cells of Rhodobacter sph- aeroides, which suggested a degree of coupling between the magnesium chelation and methyltransferase steps. This coupling was proposed to take the form of a multienzyme complex for the conversion of proto- porphyrin to magnesium protoporphyrin IX mono- methylester (MgPME) [21]. Subsequently, it was shown that when Escherichia coli cell extracts containing the magnesium chelatase H subunit of R. capsulatus (BchH) and the corresponding methyltransferase (BchM) were mixed, stimulation of BchM activity was observed [22]. However, purified ChlH from Synecho- cystis was subsequently shown to have no effect on ChlM activity [23]. These are important observations, and the significance of these findings with respect to the current data is addressed in the Discussion. In this paper we have used purified recombinant Synechocystis enzymes to demonstrate that ChlH has a dramatic stimulatory effect on ChlM catalysis. Quenched-flow experiments show that the magnesium chelatase H subunit markedly enhances ChlM catalysis by accelerating the formation and breakdown of the catalytic intermediate, providing a kinetic link between the first two reactions of chlorophyll biosynthesis, with the signalling molecule MgP as the common factor. Interactions between the methyltransferase and mag- nesium chelatase are likely to be crucial in determining the availability of MgP for both signalling [20] and biosynthetic roles in the chloroplast. Results ChlH stimulates the methyltransferase reaction ChlM (0.2 lm) was assayed in the presence of 50 lm MgD, 1 mm SAM and varying concentrations of ChlH, the porphyrin binding subunit of magnesium chelatase. MgD was used, instead of MgP, as the con- centration of this water-soluble analogue may be con- trolled more easily. Figure 1 depicts the increase in the catalytic rate of ChlM when the concentration of ChlH is increased, which implies that either a ChlH–MgD complex is acting as an activated substrate or that ChlH is directly accelerating the reaction chemistry. The plot of steady-state rate (v ss ) against ChlH concen- tration was fitted to a single rectangular hyperbola. ChlM assays were performed as previously reported [13], and v vs. [MgD] curves were obtained in the pres- ence and absence of 4 lm ChlH; this concentration of ChlH gives almost maximal stimulation of methyltrans- ferase activity. 0.2 lm ChlM was assayed with 1 mm SAM and various concentrations of MgD. Figure 2 shows the rate of MgDME evolution (lmÆmin )1 Ælm ChlM )1 ) vs. [MgD] in the presence and absence of 4 lm ChlH. Both data sets were fitted to single rectangular hyperbolae and apparent K m values were obtained. In the presence and absence of ChlH the apparent K m values were 17.3 ± 3.3 lm and 24.3 ± 7.5 lm, res- pectively. The effect of ChlH on the lag phase prior to product formation Figure 3A shows quenched-flow ChlM assays in the absence and presence of 0.75 lm ChlH. Figure 3B shows the evolution ⁄ decay of the catalytic intermediate Fig. 1. Augmentation of the methyltransferase reaction by magnes- ium chelatase H subunit (ChlH). A plot of ChlM catalytic rate (l M min )1 ÆlM ChlM )1 vs. ChlH concentration. Methyltransferase assays were performed in the presence of varying concentrations of ChlH protein. The reaction mixture contained 100 m M Tris pH 7.5, 100 m M glycerol, 0.2 lM ChlM, 20 lM MgD, 1 mM SAM and var- ious concentrations of ChlH. Error bars represent the standard errors when estimating the steady-state rate from timepoints in the stopped assay. M. Shepherd et al. Enzyme interaction in chlorophyll biosynthesis FEBS Journal 272 (2005) 4532–4539 ª 2005 FEBS 4533 in the absence and presence of 0.75 lm ChlH, and Fig. 3C depicts a typical chromatogram obtained dur- ing HPLC analysis of the quenched-flow samples. ChlM, SAM and ChlH (when present) were preincu- bated (in 100 mm Tris pH 7.5 ⁄ 100 mm NaCl) in syr- inge 1, and MgD was preincubated similarly in syringe 2. The quench solution used was the same as that in steady-state assays (acetone ⁄ H 2 O ⁄ 33% ammonia solu- tion, 80 : 20 : 1). In the absence of ChlH, the lag phase that precedes MgDME evolution is approximately 150 ms. The presence of ChlH reduces this lag phase to approximately 50 ms, and the amplitude of the burst phase is enhanced approximately fivefold. The evolution ⁄ depletion of the putative catalytic intermedi- ate was also monitored. Figure 3B demonstrates that the when ChlH is absent, the catalytic intermediate accumulates between 0 and 150 ms with a rate con- stant of 24.3 ± 4.1 s )1 . When ChlH is present, the concentration of intermediate appears to decrease immediately, suggesting that its evolution occurs on a timescale more rapid than the dead time of the instru- ment (approximately 2 ms). Hence, this process must occur with a rate constant in excess of 500 s )1 . The rate of intermediate decay (+ ChlH) was fitted to a single exponential with a rate constant of 31.7 ± 9.5 s )1 . The rate constants for product accumulation (Fig. 3A) may be estimated from the rates of inter- mediate decay (Fig. 3B); rate constants for the accumu- lation of MgDME are 11.8 s )1 (–ChlH) and 31.7 s )1 (+ 2 lm ChlH). All these parameters are summarized in Table 1. Fig. 2. Rate of ChlM catalysis vs. magnesium deuteroporphyrin IX (MgD) concentration in the presence and absence of 4 l M ChlH (Mg chelatase H subunit). Plots of ChlM catalytic rate (l MÆmin )1 per lM ChlM) vs. MgD concentration. The concentrations of ChlM and SAM were fixed at 0.2 l M, and 1 mM, respectively. Assays were performed in the presence (s) and absence (d)of4l M ChlH. K app MgD ¼ 17.3 ± 3.3 lM in the presence of ChlH. K app MgD ¼ 24.3 ± 7.5 l M in the absence of ChlH. Fig. 3. Quenched-flow ⁄ HPLC analysis of product and intermediate evolution. All solutions contained 100 m M Tris pH 7.5, and 100 mM NaCl. Immediately after mixing, concentrations of ChlM, SAM and MgD were fixed at 0.5 l M,1mM and 30 lM, respectively. This was performed when ChlM and SAM were preincubated in the pres- ence (s) and absence (d) of 0.75 l M ChlH in syringe 1. Syringe 2 contained only MgD. (A) MgDME evolution was followed by integ- rating the peaks at 12.3 min on the HPLC chromatograms for each timepoint. (B) The evolution ⁄ depletion of the putative intermediate was followed by integrating the peaks at 12.7 min on the HPLC chromatograms for each timepoint. The data for the decay of inter- mediate in the presence of ChlH were fitted to a three-parameter exponential (k ¼ 31.7 ± 9.5 s )1 ). The evolution of intermediate in the absence of ChlH was characterized by a single exponential (k ¼ 24.3 ± 4.1 s )1 ). Units are in arbitrary fluorescence units (AU). (C) A typical HPLC chromatogram to show the elution of MgD, MgDME and the catalytic intermediate (Int). Enzyme interaction in chlorophyll biosynthesis M. Shepherd et al. 4534 FEBS Journal 272 (2005) 4532–4539 ª 2005 FEBS Discussion The presence of ChlH clearly exerts a dramatic effect on the methylation of MgD catalysed by ChlM (Fig. 1). These data appear to conflict with previous work whereby purified ChlH was found to have no stimulatory effect on ChlM activity [23]. However, that study used a stopped assay where a single timepoint was taken after 30 min, which misses the much faster initial rate seen in the current study, the measurement of which is complete within 8 min. Three hypotheses present themselves: a ChlH–MgD complex is a pre- ferred substrate for the methyltransferase, ChlH binds to ChlM as an allosteric effector, or ChlH accelerates the reaction chemistry directly. The concentration dependence demonstrates that only a small excess of ChlH over ChlM is required for maximum rate enhancement (Fig. 1). The ChlH concentration at half the maximal rate is 1.2 ± 0.3 lm, which might repre- sent the binding constant (K D ) for the binding of ChlH to ChlM. When excess ChlH was present, the apparent K m MgD (K app MgD ) was 17.3 ± 3.3 lm (Fig. 2), whereas in the absence of ChlH, the K app MgD was 24.3 ± 7.5 lm (Fig. 2). The K d for ChlM binding to free MgD, deter- mined by fluorimetric titration, is 2.4 lm [13]. There- fore, if a ChlH–MgD complex is indeed a preferred substrate for ChlM, the affinity of the methyltrans- ferase for such a complex does not appear to be greater than that of free MgD. Also, given that ChlH does not significantly alter the K m MgD , these observa- tions suggest an alternative role for ChlH in stimula- ting the methyltransferase reaction. Figure 3 shows that ChlH reduces the lag phase of MgDME product evolution (Fig. 3A). This is consis- tent with the data in Fig. 3B, where the evolu- tion ⁄ depletion of the putative intermediate is monitored. When ChlH is absent, the intermediate does not reach the exponential decay phase until at least 150 ms has elapsed, which coincides with the evo- lution of MgDME [14]. When ChlH is present, the exponential decay phase of the intermediate occurs much earlier (Fig. 3B), and intermediate accumulation occurs within the 2 ms dead time of the instrument. This dramatic acceleration in formation of the interme- diate by ChlH, as well as reduction in its lifetime, is consistent with the concomitant decrease in lag phase of product evolution in Fig. 3A. The accumulation of intermediate in the absence of ChlH was fitted to an exponential with a rate constant of 24.3 ± 4.1 s )1 , which compares to 11.9 s )1 with previous work [14]. The current value is a better estimate of intermediate accumulation, as the fit in Fig. 3B considers only the evolution of intermediate. The rate constant for the decay of intermediate in the presence of ChlH (31.7 ± 9.5 s )1 ) is three times as large as the value recorded in the absence of ChlH (11.8 s )1 [14]). These rate constants can be used to estimate the rates of MgDME accumulation in Fig. 3A, which suggests that ChlH elicits a threefold increase in the rate of product accumulation (Table 1). These data demonstrate that ChlH enhances both the accumulation and decay of this reaction intermediate, resulting in a reduction in the lag phase of product accumulation, and an increase in initial rate of product evolution. Furthermore, the presence of ChlH increases the magnitude of the burst phase approximately fivefold (Table 1), which implies that a greater concentration of enzyme is available to bind MgDME. As ChlM appears to bind the inter- mediate more transiently, this is likely to yield a higher available concentration of ChlM to bind other mole- cular species in the reaction. ChlH appears to enhance catalysis by accelerating the formation and decay of a catalytic intermediate. This is depicted in Scheme 1. One cannot yet pinpoint the exact mode of action of ChlH, although it is possible that ChlH may possess reactive sidechains involved in methyltransferase catalysis. Such roles may include the stabilization of the positive charge on the methyl carbon of SAM, or the enhancement of the negative charge on the propionate carboxyl groups of MgD. The rate constants quoted in Scheme 1 are all Table 1. Summary of kinetic parameters for Synechocystis ChlM, and the effects of the magnesium chelatase ChlH subunit (rate constants refer to Scheme 1). Parameter ChlM ChlM + ChlH K app MgD 24.3 ± 7.5 lM 17.3 ± 3.3 lM Rate constant for formation of catalytic intermediate 24.3 ± 4.1 s )1 (k 4 )>500s )1 (k 6 ) Rate constant for decay of intermediate 11.8 s )1 s )1 (k 5 ) [14] 31.7 ± 9.5 (k 7 ) Lag phase preceding MgDME product formation 50 ms 150 ms Magnitude of burst in product formation Approx. 10 n M Approx. 50 nM Rate constant for MgDME product formation a 11.8 s )1 31.7 s )1 [14] a Rate constants for MgDME product formation have been estimated from the rates of intermediate decay. M. Shepherd et al. Enzyme interaction in chlorophyll biosynthesis FEBS Journal 272 (2005) 4532–4539 ª 2005 FEBS 4535 faster than k cat . It has previously been proposed that product release is the slow step in the reaction [14]. This is consistent with the current work, as ChlH does not enhance k cat . A recent study showed that MgP accumulation triggered the alleviation of repression of photosyn- thetic genes in Arabidopsis [20], and MgP is suggested to be a signal for one of the plastid to nucleus signal- ling pathways. However, the relative catalytic rates of magnesium chelatase and MgP methyltransferase may dictate that there is very little free MgP available for signalling. We have estimated that k cat for Mg chela- tion is 0.8 min )1 [24], whereas k cat for the subsequent methyltransferase step is estimated to be 3.4 min )1 , and this is in the absence of ChlH [14]. This implies that in vivo, there may be little unbound MgP, especi- ally when ChlH is present in excess over that required for Mg chelation, since methyltransferase activity will be greatly stimulated. The discovery that another pro- tein, Gun4, can both stimulate Mg chelatase and bind MgP [12,25] adds another layer of complexity to both the regulation of chlorophyll biosynthesis, and the availability of MgP for signalling to the nucleus. We suggest that the relative amounts of both Gun4 and CHLH are crucial factors that regulate both flux down the early part of the chlorophyll biosynthetic pathway and the availability of the MgP signalling molecule. It is known that CHLH expression exhib- its diurnal fluctuations in Antirrhinum, Arabidopsis, barley and soybean [3,26–28] and that CHLH is regu- lated by a circadian clock [29]. A regulatory mecha- nism whereby alterations in magnesium chelatase H subunit levels affect partitioning between the mag- nesium (chlorophyll) and iron (haem) branches of tetrapyrrole biosynthesis was proposed by Gibson et al. [26]. Our quantitative data reported here extend the influence of ChlH and show for the first time the way in which this protein exerts a strong effect on the next enzyme in the pathway, ChlM. Inspection of the ChlH titration in Fig. 1 leads to the conclusion that temporal variations in CHLH (ChlH in plants) concentration in vivo may greatly influence the cata- lytic rate of CHLM, the eukaryotic MgP methyl- transferase. This has important implications for the coupling between these steps and for the availability of MgP for signalling. Scheme 2 summarizes these conclusions in terms of variation the magnesium che- latase H subunit and its effect on the tetrapyrrole branchpoint and ChlM, but neglects the effect of Gun4. The fact that variations in ChlM activity are accompanied by altered ferrochelatase activity has been shown recently using transgenic approaches [30]. It would be necessary to establish the levels of these proteins in vivo in order to apply the enhancements measured in this study to a more physiologically rele- vant situation. Experimental procedures All pigments were purchased from Porphyrin Products (Logan, UT, USA). The remaining chemicals were pur- chased from Sigma-Aldrich unless otherwise specified. Protein expression and purification The plasmid pET9a-His 6 -ChlM [23] was transformed into E. coli BL21 (DE3) cells and the Synechocystis chlM gene was induced for 15 h at 20 °C using 0.4 mm isopropyl Scheme 1. The ChlM reaction and the proposed involvement of the magnesium chelatase H subunit. The rate constants are described in Table 1. ChlM, ChlH and the catalytic intermediate are abbreviated as E, H, and Int, respectively. Enzyme interaction in chlorophyll biosynthesis M. Shepherd et al. 4536 FEBS Journal 272 (2005) 4532–4539 ª 2005 FEBS thio-b-d-galactoside. The cells were harvested at 3000 g at 4 °C and cells from 2 L of culture were resuspended in 20 mL chilled binding buffer [20 mm citrate ⁄ KOH (pH 5.8), 500 mm NaCl, 500 mm glycerol, 5 mm imidazole]. The cells were disrupted by sonication for 6 · 30 s on ice, and the cell debris was removed at 39 000 g at 4 °C. The supernatant was loaded at 2 mLÆmin )1 onto a 2.0 cm · 5.0 cm column packed with Chelating Sepharose Fast-Flow resin (Amersham Biosciences, Uppsala, Sweden) charged with 50 mm NiSO 4 and pre-equilibrated with three column volumes of binding buffer. The column was washed with 10 column volumes of binding buffer and 6 column vol- umes of binding buffer containing 60 mm imidazole (wash buffer) to remove any loosely bound contaminants. The His-tagged ChlM was eluted with binding buffer containing 250 mm imidazole (elute buffer). A 50 mL column of P-6 desalting gel (Bio-Rad, Hercules, CA, USA) was equili- brated with 50 mm citrate ⁄ KOH (pH 5.8), 300 mm gly- cerol, 200 mm NaCl and used to remove imidazole from the buffer. A typical yield was 15 mg protein from a 2 L culture of E. coli. Porphyrin stocks Porphyrin solutions were freshly prepared by dissolving a small amount of porphyrin in buffer. A more water-soluble analogue, magnesium deuteroporphyrin (MgD) was used instead of MgP. The presence of detergent in the assay buf- fer was no longer required. Porphyrin concentrations were determined in 0.1 m HCl using the e 398 of 433 000 m )1 Æcm )1 [31] after Mg 2+ had been removed from the porphyrin by a 5-min incubation in 1 m acetic acid. SAM and S-adenosyl- l-homocysteine (SAH) stock solutions were prepared daily in 0.1 m HCl and 0.1 m NaOH, respectively. Their concen- trations were determined using the e 256 of 15 200 m )1 Æcm )1 in 1 m HCl for SAM and e 260 of 16 000 m )1 Æcm )1 at pH 7 for SAH [32]. ChlM assays Reactions were carried out at 30 °C in 100 mm Tris pH 7.5, 100 mm glycerol, 0.2 lm ChlM, and MgD and SAM concentrations as indicated in the figure legends. The assay mixtures were incubated at 30 °C in the absence of SAM for 5 min to allow for thermal equilibration. The SAM was added, and 20-lL aliquots were taken every 2 min over a period of 8 min and quenched in 400 lL stop solution (acetone ⁄ water ⁄ 33% ammonia solution, 80 : 20 : 1). These aliquots were centrifuged at 20 000 g for 5 min to pellet any aggregated protein. Pigments were sep- arated using reversed phase HPLC [13,14]. Between 10 and 70 lL of soluble phase, depending on the MgD concentra- tion, was loaded onto a Beckman ODS Ultrasphere column (150 · 4.6 mm; CA, USA). The pigments were separated by a 7-min linear gradient from 0% to 67% solvent B at 2mLÆmin )1 , and then the gradient was paused for a further 5 min for the porphyrins to elute (Solvent A ¼ 0.005% tri- ethylamine in water, solvent B ¼ acetonitrile). Eluted por- phyrins were detected with a Waters in-line fluorescence detector. Excitation and emission wavelengths were 394 ± 5 nm and 580 ± 5 nm, respectively. The peaks that corresponded to MgD obtained in the elution profiles were integrated using Waters Millennium software. Known amounts of MgDME were analysed in the same way to produce a standard curve. The maximum rate during an assay was taken as the steady-state rate and occurred at the beginning of the reaction. Quenched-flow measurements Pre-steady state time samples from ChlM-catalysed reac- tions were obtained using a Hi-Tech rapid quenched flow system. All solutions contained 100 mm Tris pH 7.5, and 100 mm NaCl. The reaction cell was maintained at a con- Scheme 2. Diagram of the branchpoint of tetrapyrrole biosynthesis, showing the parti- cipation of the Mg chelatase H subunit in both the chelatase and methyltransferase steps. The K D for the H ⁄ ID interaction was taken from Jensen et al. 1998 [24], and the K D for the association with ChlM is from Fig. 1. The possible effects of varying the concentration of the magnesium chelatase H subunit in the 0.2–0.5 l M range are also presented, although the effects of Gun4 or varying porphyrin concentrations are not included. M. Shepherd et al. Enzyme interaction in chlorophyll biosynthesis FEBS Journal 272 (2005) 4532–4539 ª 2005 FEBS 4537 stant temperature of 30 °C by circulation of water from a thermostatically controlled water bath (Grant Instruments, Cambridge, UK). Reactions were quenched in stop solution (acetone ⁄ water ⁄ 33% ammonia solution, 80 : 20 : 1, v ⁄ v ⁄ v), and the concentrations of MgDME and intermediate were determined using reversed phase HPLC, as previously described [13,14]. 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Enzyme interaction in chlorophyll biosynthesis FEBS Journal 272 (2005) 4532–4539 ª 2005 FEBS 4539 . Kinetic basis for linking the first two enzymes of chlorophyll biosynthesis Mark Shepherd, Samantha McLean and C. Neil Hunter Robert Hill Institute for. accelerating the formation and breakdown of the catalytic intermediate, providing a kinetic link between the first two reactions of chlorophyll biosynthesis, with the

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