Kineticbasisforlinkingthefirsttwoenzymes 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 ofthe 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 ofthe 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. Thekinetic coupling between the chelatase and methyltrans-
ferase has important implications for regulation ofchlorophyll biosynthesis
and forthe 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) forthe ChlM catalysed reaction have been
implicated in plastid-to-nucleus signalling [15–20]. As
MgP is also the product ofthe magnesium chelatase
reaction, this emphasizes the importance of quantita-
tive studies not only ofthe methyltransferase and
chelatase, but also ofthe interaction between these
enzymes. The coupling ofthe 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 forthe 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 firsttwo reactions ofchlorophyll 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 ofthe catalytic intermediate
Fig. 1. Augmentation ofthe 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 ofthe 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 ofthe 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 ofthe 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 forthe 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 ofthe putative intermediate
was followed by integrating the peaks at 12.7 min on the HPLC
chromatograms for each timepoint. The data forthe 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 chlorophyllbiosynthesis 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 forthe 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
) forthe 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 ofthe 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 ofthe 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 ofthe intermediate occurs
much earlier (Fig. 3B), and intermediate accumulation
occurs within the 2 ms dead time ofthe instrument.
This dramatic acceleration in formation ofthe 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 ofthe 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 ofthe 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 ofkinetic parameters for Synechocystis ChlM, and the effects ofthe 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 ofthe 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 ofchlorophyll 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 ofthechlorophyll biosynthetic
pathway and the availability ofthe 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 forthefirst 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 forthe 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 ofthe 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 chlorophyllbiosynthesis 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 forthe 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 ofthe 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 ofthe branchpoint of
tetrapyrrole biosynthesis, showing the parti-
cipation ofthe 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 ofthe 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]. The data obtained was analysed using
nonlinear regression (sigmaplot 8.0), and apparent rate
constants were obtained by fitting the plots to a single
exponential [y ¼ y0 + (1-e
–bx
)].
Acknowledgements
We thank Mark Hoggins for assisting with the data
analysis. This research was funded by the Biotechno-
logy and Biological Sciences Research Council, UK.
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M. Shepherd et al. 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