Regulation of ribulose-1,5-bisphosphate carboxylase/oxygenase (Rubisco) activase: Product inhibition, cooperativity, and magnesium activation

Article abstract of DOI:10.1074/jbc.M115.651745

In many photosynthetic organisms, tight-binding Rubisco inhibitors are released by the motor protein Rubisco activase (Rca). In higher plants, Rca plays a pivotal role in regulating CO₂ fixation. Here, the ATPase activity of 0.005 mM tobacco Rca was monitored under steady-state conditions, and global curve fitting was utilized to extract kinetic constants. The k_cat was best fit by 22.3 ± 4.9 min^-1, the K_m for ATP by 0.104 ± 0.024 mM, and the K_i for ADP by 0.037 ± 0.007 mM. Without ADP, the Hill coefficient for ATP hydrolysis was extracted to be 1.0 ± 0.1, indicating noncooperative behavior of homo-oligomeric Rca assemblies. However, the addition of ADP was shown to introduce positive cooperativity between two or more subunits (Hill coefficient 1.9 ± 0.2), allowing for regulation via the prevailing ATP/ADP ratio. ADP-mediated activation was not observed, although larger amounts led to competitive product inhibition of hydrolytic activity. The catalytic efficiency increased 8.4-fold upon cooperative binding of a second magnesium ion (Hill coefficient 2.5 ± 0.5), suggesting at least three conformational states (ATP-bound, ADP-bound, and empty) within assemblies containing an average of about six subunits. The addition of excess Rubisco (24:1, L8S8/Rca6) and crowding agents did not modify catalytic rates. However, high magnesium provided for thermal Rca stabilization. We propose that magnesium mediates the formation of closed hexameric toroids capable of high turnover rates and amenable to allosteric regulation. We suggest that in vivo, the Rca hydrolytic activity is tuned by fluctuating [Mg²⁺] in response to changes in available light.

Full text of DOI:10.1074/jbc.M115.651745

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THE JOURNAL OF BIOLOGICAL CHEMISTRY VOL. 290, NO. 40, pp. 24222–24236, October 2, 2015
© 2015 by The American Society for Biochemistry and Molecular Biology, Inc. Published in the U.S.A.
Regulation of Ribulose-1,5-bisphosphate Carboxylase/
Oxygenase (Rubisco) Activase
PRODUCT INHIBITION, COOPERATIVITY, AND MAGNESIUM ACTIVATION*
Received for publication, March 12, 2015, and in revised form, August 4, 2015 Published, JBC Papers in Press, August 17, 2015, DOI 10.1074/jbc.M115.651745
Suratna Hazra, J. Nathan Henderson, Kevin Liles, Matthew T. Hilton, and Rebekka M. Wachter1
From the Department of Chemistry and Biochemistry and Center for Bioenergy and Photosynthesis, Arizona State University,
Tempe, Arizona 85287
Background: Rubisco activase (Rca) maintains carbon fixation, coordinates photosynthetic pathways, and regulates dark-
light transitions.
Results: Positive cooperativity of ATP hydrolysis is afforded by ADP binding to oligomeric assemblies in the Rca-Mg-ATP-Mg
state.
Conclusion: Magnesium-mediated allosteric regulation involves the coexistence of ATP- and ADP-bound states.
Significance: In dark-adapted chloroplasts, wasteful ATP hydrolysis may be prevented by low magnesium.
In many photosynthetic organisms, tight-binding Rubisco
inhibitors are released by the motor protein Rubisco activase
(Rca). In higher plants, Rca plays a pivotal role in regulating CO2
fixation. Here, the ATPase activity of 0.005 mM tobacco Rca was
monitored under steady-state conditions, and global curve fit-
ting was utilized to extract kinetic constants. The kcat was best fit
by 22.3 ؎ 4.9 min؊1, the Km for ATP by 0.104 ؎ 0.024 mM, and
the Ki for ADP by 0.037 ؎ 0.007 mM. Without ADP, the Hill
coefficient for ATP hydrolysis was extracted to be 1.0 ؎ 0.1,
indicating noncooperative behavior of homo-oligomeric Rca
assemblies. However, the addition of ADP was shown to intro-
duce positive cooperativity between two or more subunits (Hill
coefficient 1.9 ؎ 0.2), allowing for regulation via the prevailing
ATP/ADP ratio. ADP-mediated activation was not observed,
although larger amounts led to competitive product inhibition
of hydrolytic activity. The catalytic efficiency increased 8.4-fold
upon cooperative binding of a second magnesium ion (Hill coef-
ficient 2.5 ؎ 0.5), suggesting at least three conformational states
(ATP-bound, ADP-bound, and empty) within assemblies con-
taining an average of about six subunits. The addition of excess
Rubisco (24:1, L8S8/Rca6) and crowding agents did not modify
catalytic rates. However, high magnesium provided for thermal
Rca stabilization. We propose that magnesium mediates the for-
mation of closed hexameric toroids capable of high turnover
rates and amenable to allosteric regulation. We suggest that in
vivo, the Rca hydrolytic activity is tuned by fluctuating [Mg2؉] in
response to changes in available light.
tic action of Rubisco activase (Rca) to cope with a variety of
inhibitory mechanisms. Some of these are regulatory by nature,
such as nocturnal inhibition (1) or inhibition of decarbamylated
Rubisco by its substrate ribulose 1,5-bisphosphate (RuBP) (2),
whereas others appear to be intrinsic to turnover in an oxygenic
atmosphere (3, 4). Regardless, the release of competitive inhib-
itors is substantially accelerated by the motor protein Rca, with
subsequent degradation of some inhibitors by their specific
phosphatases (5, 6). A comprehensive understanding of all co-
regulatory mechanisms that enhance or diminish biological
carbon fixation is critical in predictive modeling of net photo-
synthesis and biological pathway engineering to improve plant
performance (7–9).
In higher plants, Rca plays a key role in modulating the over-
all level of carbon fixation, as well as the rate of photosynthetic
induction under changing environmental conditions (10). With
the aid of ATP hydrolysis, hexameric ring-like assemblies of
Rca are thought to shift the conformational equilibrium of
Rubisco to favor dissociation of dead-end inhibitors from the
active sites. Within the chloroplast stroma, the light-dependent
energy charge is thought to control Rca activity through the
prevailing ATP/ADP ratio and redox status (10). Many plants
express Rca in two isoforms. The longer ␣-isoform is distin-
guished from the shorter ␤-isoform by the presence of a 30-res-
idue C-terminal extension that harbors two conserved redox-
active cysteine residues regulated via the thioredoxin-f system
(11). The Rca subunit concentration in the chloroplast stroma
has been estimated to be ϳ7 to ϳ21 mg/ml (160–480 ␮M) (12),
whereas the Rubisco holoenzyme concentration is ϳ500 ␮M.
These values suggest a ratio of approximately 1 Rca hexamer
per 10 Rubisco holoenzymes, crowded together in an environ-
ment where ϳ3% of all stromal proteins consist of Rca and
ϳ40% of Rubisco.
The primary carbon-fixing enzyme ribulose-1,5-bisphos-
phate carboxylase/oxygenase (Rubisco)2 requires the synergis-
* This work was supported by United States Department of Energy, Office of
Basic Energy Sciences, Photosynthetic Systems Grant DE-FG02–09-
ER16123 (to R. M. W). The authors declare that they have no conflicts of
interest with the contents of this article.
Multisubunit Assemblies of Rca—Rca is a member of the
ϩ
AAA superfamily of P-loop ATPases (13, 14). Frequently,
1 To whom correspondence should be addressed. Tel.: 480-965-8188; Fax:
480-965-2747; E-mail: rwachter@asu.edu.
2 The abbreviations used are: Rubisco, ribulose-1,5-bisphosphate carboxyl-
ase/oxygenase; Rca, Rubisco activase; RuBP, ribulose 1,5-bisphosphate;
ATP␥S, adenosine 5Ј-O-[␥-thio]triphosphate); PNP, purine nucleoside
phosphorylase; MESG, 2-amino-6-mercapto-7-methylpurine ribonucleo-
side; Tricine, N-[2-hydroxy-1,1-bis(hydroxymethyl)ethyl]glycine; ER, endo-
plasmic reticulum; ECM, extracellular matrix.
24222 JOURNAL OF BIOLOGICAL CHEMISTRY
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Steady-state Turnover of Rubisco Activase
ϩ
proper assembly of AAA proteins requires nucleotide binding chains, the tobacco structure exhibits a shorter pitch along the
(15–18), and some members utilize their partner proteins as helical axis (32), whereas the Arabidopsis structure exhibits a
ϩ
ϩ
assembly scaffolds (19). Although AAA hexameric rings tighter hinge-bending angle between the AAA N- and C-do-
appear to be most common (18, 20–23), some exceptions to mains (39). This subtle movement is likely due to the trapping
this rule are known (24–26). In general, higher plant Rca prep- of a sulfate ion in the nucleotide-binding pocket. Structural
ϩ
arations are highly polydisperse in vitro. Recently, we have pro- information for the N- and C-terminal extensions of the AAA
vided experimental evidence that in vitro the hexamer forma- domain is not yet available.
ϩ
tion of cotton ␤-Rca is maximized around 30 ␮M subunit
Within the classic clade, many AAA proteins control pro-
concentration (27), with close to 80% of protomers forming tease activity, such as the bacterial proteins HslU, FtsH, ClpA,
six-subunit assemblies in the presence of excess ATP␥S and ClpX, and the archaeal proteasomal PAN ATPase. Most closely
high magnesium ion concentrations (28). These data suggest related to Rca are the ClpA D2 domain (15% sequence identity
ϩ
that in the chloroplast stroma, Rca is always fully assembled to with the tobacco ␤-Rca AAA module), FtsH (14%), and ClpX
a hexameric or higher order form.
(10%). For some of these proteins, x-ray structures of both spi-
Recently, it has become clear that Rca proteins derived from ral assemblies (40) and topologically closed 6-unit rings have
different plant species show differences in their in vitro assem- been reported (41, 42). For a subset of these such as HslU, both
bly mechanism. For example, the assembly of tobacco ␤-Rca 6-fold symmetric and asymmetric closed rings have been
was reported to be nucleotide-independent (29). In addition, a described (15, 43). Notably, 6-fold (41), 2-fold (42), and 3-fold
subunit concentration of 5 ␮M (0.2 mg/ml) or larger was shown (44) crystal symmetries were observed for FtsH. For all these
to provide a weight-averaged sedimentation coefficient consis- systems, it remains unclear which of the available hexameric
tent with a hexamer (29). However, these experiments also arrangements is most relevant to the catalytic cycle.
demonstrated that tobacco ␤-Rca remained highly polydis-
ATPase Activity and Rubisco Reactivation Models—Rca is
perse over the entire concentration range tested (30). The sta- capable of hydrolyzing ATP both in the presence and in the
ble formation of a hexameric species could only be demonstrated absence of Rubisco, and the addition of small amounts of
for the tobacco ␤-Rca-R294V mutant (31, 32). By negative stain Rubisco to the reaction mixture has been shown to have no
electron microscopy, this mutant provided images of closed-ring effect on the hydrolytic rate (12, 32). Typically, published
hexamers in the presence of either ATP⅐Mg or ATP␥S⅐Mg (32).
kinetic constants have been estimated using Rca subunit con-
Some years ago, spinach ␣-Rca was shown to exhibit substan- centrations at or below 2.3 ␮M (0.10 mg/ml), with a recent
tially elevated thermal stability in the presence of ATP␥S⅐Mg report indicating 48 turnovers/sϪ1 for tobacco ␤-Rca (30). At 2
(33). This complex was recently reported to form obligate hexa- ␮M Rca, turnover rates have been reported to range from 45 to
meric species in vitro, in stark contrast to the shorter spinach 60 minϪ1 in the presence of 2 and 2.5 mM ATP, respectively (32,
␤-Rca, which was reported to exhibit a high degree of size het- 45). For other species, reported rates were 30.4 minϪ1 for spin-
erogeneity under identical conditions (29). Spinach ␤-Rca ach ␤-Rca at 2 mM ATP (33), 17.4 minϪ1 for cotton ␤-Rca at 4
appeared to aggregate beyond the hexameric state, even in the mM ATP (46), and 23.9 and 32.6 minϪ1 for Arabidopsis ␤-Rca at
presence of excess ATP␥S⅐Mg (29). This behavior is more sim- 2 and 4 mM ATP, respectively, measured by two different assay
ilar to that reported for other higher plant Rca proteins, such as types (47). Utilizing fluorescence quenching of a protein-bind-
creosote, tobacco, and Arabidopsis Rca (34, 35).
ing dye, Kd values of 0.57 and 5.9 ␮M were reported for ADP⅐Mg
Structural Organization of Rca Subunits and Relationship to and ATP⅐Mg, respectively (45). A 4-min end point assay
ϩ
ϩ
the AAA Superfamily—The 200–250-residue AAA module revealed loss of activity of Arabidopsis ␤-Rca in the presence of
Ϫ1
contains an ATP-binding cassette with an N-terminal domain ADP. Apparently, the enzyme turned over 45.6 min in the
Ϫ1
harboring the Walker A and B motifs. The C-terminal domain presence of ATP alone, 25.2 min at a molar ratio of ADP/
is connected by a linker, such that a nucleotide-binding pocket is ATP equal to 1:3, and 7.8 minϪ1 at a molar ratio of ADP/ATP
formed at the hinge region. Oligomerization allows a conserved equal to 1 (11). Similarly, the ATPase activity of spinach and
Arg of the N-domain (“Arg finger”) to contact the ATP ␥-phos- cotton ␤-Rca was demonstrated to be 20% of maximum at an
phate bound to the adjacent subunit (36). In this way, conforma- ADP/ATP equal to 1:3 (46). In the same work, the cotton ␤-Rca
tional adjustments are likely communicated between subunits, in ATPase activity was measured as a function of Rca concentra-
step with catalytic cycles of ATP binding, hydrolysis, and ADP tion up to 1.8 ␮M (0.080 mg/ml), indicating a relatively steady
release. If the system is loaded with a cognate macromolecule, increase in hydrolytic rate up to 13 turnovers minϪ1 (46).
mechanical work will lead to the structural remodeling of the tar-
get protein.
Although several Rca-Rubisco binding geometries have been
proposed (9, 32, 48), the actual mechanisms of recognition,
Currently, x-ray structures are available for bacterial Rca (37) interaction, and activation of Rubisco remain unknown. It is
and for higher plant Rca proteins from two different species. tempting to speculate that the sequestration of less ordered
The 1.9 Å resolution creosote bush substrate recognition Rubisco segments by the central pore of Rca toroids plays a role,
ϩ
domain (38) consists of a helical bundle that forms the core of as demonstrated for a number of other AAA assemblies, such
ϩ
the AAA C-domain and an insertion that bears the specificity as FtsH (42), PAN (49), ClpX (50), Cdc48 (51), and ClpA/B (18).
residues. The 2.9 Å resolution structures of the nucleotide-free A mutational analysis of tobacco ␤-Rca pore loop residues has
ϩ
AAA modules of tobacco (32) and Arabidopsis thaliana (39) provided evidence consistent with a peptide binding or thread-
Rca demonstrate a spiral arrangement of subunits in the crystal. ing mechanism (32). Regardless, a significant difficulty in devis-
Although in both structures, one turn consists of six protein ing binding models is imposed by the poorly understood extent
OCTOBER 2, 2015•VOLUME 290•NUMBER 40
JOURNAL OF BIOLOGICAL CHEMISTRY 24223

Steady-state Turnover of Rubisco Activase
ϩ
of Rca shape modification upon nucleotide hydrolysis. NADH to NAD (52). The reaction mixture contained 100 mM
Although such structural adjustments would provide the Tricine, pH 8.0, 20 mM KCl, 5 mM DTT, 5 mM MgCl2, 2 mM
mechanical force to open Rubisco active sites, the process itself phosphoenolpyruvate, 0.3 mM NADH, 4.8–8 units of pyruvate
may or may not involve direct interactions with these sites.
kinase, and 7.2–11.2 units of lactate dehydrogenase (Sigma).
To date, key features of the Rca ATPase activity remain Upon addition of an aliquot of Rca to a cuvette containing assay
poorly understood, such as the allosteric control mechanisms mixture to provide a final volume of 1.0 ml, the A340 was mon-
of multisubunit assemblies thought to fine-tune hydrolytic itored for 60 s to obtain a stable signal. Subsequently, ATP was
activity. In work presented here, steady-state turnover kinetic added, the contents were mixed manually with a cuvette mixer,
methods were utilized to investigate the regulation of Rca and the reaction was monitored for 300 s. The extinction coef-
Ϫ1
ATPase activity by the available adenine pool, by magnesium ficient of NADH (6,220 M cmϪ1) was used to calculate the
binding, and by interactions with Rubisco. This work provides a rate of NADH loss.
significant step toward the quantitative description of ADP
EnzChek Phosphate Assay (MESG Assay)—The rate of inor-
inhibition as product concentrations rise. We demonstrate ganic phosphate release due to the ATPase activity of Rca was
subunit communication between at least three protomers, monitored by an enzyme-coupled spectrophotometric assay
mediated by magnesium binding that augments catalytic activ- (53). In this assay, purine nucleoside phosphorylase (PNP) is uti-
ity 8–9-fold. We anticipate that steady-state kinetic data will lized to catalyze the reaction of phosphate with the guanosine ana-
form the foundation for the development of models for the log 2-amino-6-mercapto-7-methylpurine ribonucleoside (meth-
reaction cycle in terms of stochastic, coordinated sequential ylthioguanosine MESG, ␭
330 nm), which generates the
max
switching or concerted conformational switching. Ultimately, corresponding purine product 2-amino-6-mercapto-7-methylpu-
the elucidation of the Rca ATPase mechanism is expected to rine (␭ 360 nm) and ribose 1-phosphate. To assay inorganic
max
help answer critical questions in bioenergetics, such as ATP phosphate production by Rca in a continuous fashion, the absor-
consumption per Rubisco reactivation event.
bance difference between the PNP reactant and product species
was monitored at 360 nm. The assay is quantitative between 2 and
150␮M phosphateandbetweenpH6.5and8.5. Theprocedurewas
Materials and Methods
Preparation of Cotton and Tobacco Short Form (␤) Rca—Cot- modified from the manufacturer’s recommendations (Molecular
ton ␤-Rca bearing a C-terminal Ala-Cys insert and tobacco Rca Probes, Eugene, OR) by increasing the amount of PNP 3-fold and
were cloned into the pET151/D-TOPO vector and transformed by addition of various buffer components, as described in detail
into Escherichia coli BL21*(DE3) (Invitrogen) as described pre- below.
viously (27). This procedure results in a construct bearing an
To prepare a 1 mM stock solution, MESG as provided by the
N-terminal His6 tag followed by a tobacco etch virus protease manufacturer was solubilized in 20 ml of Nanopure water. To pre-
cleavage site. Liquid cultures were grown, and protein was puri- pare PNP stock solution, the enzyme as provided by the manufac-
fied essentially as described previously (35). Briefly, bacterial turer was solubilized in 500 ␮l of Nanopure water. An ATP stock
cells were grown in 3 liters of culture to A600 ϳ0.6, then induced solution was prepared fresh each day and consisted of 500 mM
by addition of isopropyl 1-thio-␤-D-galactopyranoside, and ATP (purity Ͼ99%, Sigma) in 100 mM HEPES, pH 8.0.
grown at 25 °C and 180 rpm for 8 h. The cells were pelleted,
Preparation of the EnzCheck Standard Curve—To generate a
stored at Ϫ80 °C, and lysed by sonication in the presence of 0.5 phosphate standard curve, a standard curve working solution
mM ADP. Purification entailed affinity chromatography using was prepared by combining 550 ␮l of 1 M HEPES, pH 8.0, 500 ␮l
nickel-nitrilotriacetic acid resin in the presence of 0.5 mM ADP, of 20ϫ assay kit buffer (50 mM Tris, 1 mM MgCl2, 0.1 mM
cleavage of the His6 tag with tobacco etch virus protease, fol- sodium azide), 16 ␮l of 500 mM DTT, 80 ␮l of 500 mM MgCl2,
lowed by repurification over nickel-nitrilotriacetic acid. Protein and 6.254 ml of Nanopure water (7.4 ml final volume). 740 ␮l of
fractions were concentrated and buffer-exchanged using a working solution were added to the sample and reference
PD-10 column (GE Healthcare). The final buffer consisted of 25 cuvettes, and the A360 was set to 0. Subsequently, between 10
mM HEPES, pH 8.0, 300 mM KCl, 10% glycerol, 1 mM DTT, with and 29 ␮l of nanopure water, 200 ␮l of MESG stock, and 30 ␮l
ADP added as required for the particular experiment, usually 2 of PNP stock (3 units) were added to the sample cuvette, and the
mM ADP. Protein aliquots were frozen in liquid N2 and stored time scan was started. After 60 s, either 1 ␮l of water blank or
at Ϫ80 °C. Apo-Rca was prepared by omitting ADP from all between 1 and 20 ␮l of 5 mM potassium phosphate were added
buffers used in purification and storage. Protein was quanti- (final 5-100 ␮M Pi), such that the combined addition of water
tated utilizing the Bradford method with BSA as a standard.
and phosphate equaled 30 ␮l. The solution (1.0 ml total vol-
Nucleotides—ADP (97% pure) was purchased from Alfa ume) was mixed using a manual cuvette mixer, and the A360 was
Aesar, and ultrapure ATP (Ն99%) was purchased from Sigma monitored for 300 s. For each scan, the maximum absorbance
or Alpha Aesar. The NADH assay was utilized to determine upon signal saturation was corrected by subtracting the absor-
that ultrapure ATP (Ն99% Sigma or Alpha Aesar) contained bance observed prior to phosphate addition. The A360 values
0.36–0.42% ADP contamination.
were plotted as a function of phosphate concentration and fit to
NADH Activity Assay—The rate of ATP hydrolysis was mea- a straight line by linear regression.
sured by monitoring the rate of ADP release utilizing an
EnzCheck Baseline Scan in the Absence of Rca and ATP—To
enzyme-coupled continuous spectrophotometric assay. In this collect blank runs and to assay for Rca activity, 7.00 ml of turn-
assay, the coupled actions of pyruvate kinase and lactate dehy- over working solution was prepared by combining 550 ␮l of 1 M
drogenase provide for stoichiometric use of ADP to convert HEPES, pH 8.0, 500 ␮l of 20ϫ assay kit buffer, 16 ␮l of 500 mM
24224 JOURNAL OF BIOLOGICAL CHEMISTRY
VOLUME 290•NUMBER 40•OCTOBER 2, 2015

Steady-state Turnover of Rubisco Activase
DTT, and 5.934 ml of Nanopure water. To collect a blank absor- to the pool of ATP. This second equilibrium was not included in
bance time scan in the absence of both Rca and ATP, 700 ␮l of the calculations of free cation concentrations, because the protein-
turnover working solution were added to each sample and ref- based second Mg2ϩ-binding site may involve coordination to ATP
erence cuvettes; the A360 was autozeroed, 200 ␮l of MESG as well.
stock, 30 ␮l of PNP stock, 12 ␮l of 0.67 M MgCl2 stock, and 50 ␮l
The ATPase assays were repeated with ATP concentrations
of Rca buffer blank were added to the sample cuvette, and the between 0 and 4 mM, and concomitant adjustment of total mag-
time scan was started. After 60 s, 8 ␮l of HEPES, pH 8.0, were nesium was based on the desired conditions. Initial velocities
added, the solution was mixed, and the A360 was monitored for were converted to initial turnover rates ko (minϪ1), and com-
300 s. These conditions provide for a total Mg2ϩ concentration puter-fitted to a steady-state turnover model using the pro-
of 9.0 mM in a 1-ml final volume in the cuvette (1 mM Mg2ϩ grams KaleidagraphTM or OriginTM (Equation 1). In the fitting
contributed by the assay kit buffer).
procedure, nH is the Hill coefficient, kcat the maximal turnover
EnzCheck Baseline Scan to Monitor Residual Phosphate—To rate upon substrate saturation, and Km the Michaelis constant.
control for residual phosphate present in the assay compo-
nH
k
cat ϫ ͓S͔
nents, a baseline was collected in the presence of ATP but with-
out Rca. As before, 700 ␮l of turnover working solution were
added to each of the samples and reference cuvette; the A360
was autozeroed, and 200 ␮l of MESG stock, 30 ␮l of PNP stock,
12 ␮l of 0.67 M MgCl2 stock, and 50 ␮l of Rca buffer blank were
added to the sample cuvette, and the time scan was started.
After 60 s, 8 ␮l of 500 mM ATP stock was added to provide a
final concentration of 4 mM ATP in a 1.00-ml volume. The solu-
tion was mixed with a manual cuvette mixer, and the A360 was
monitored for 300 s. For different experiments, the volumes of
MgCl2 and ATP stock solutions were adjusted as needed, while
keeping the total volume of the 2 aliquots equal to 20 ␮l.
ko ϭ
(Eq. 1)
nH
km ϩ ͓S͔
EnzCheck ADP Inhibition Studies—The EnzCheck assay
described above was adapted to measure initial rates of ATPase
activity in the presence of varying concentrations of ADP, while
keeping the “free” magnesium ion concentration constant at 5.0
mM. The magnesium ion concentration in the turnover work-
ing solutions was adjusted to give the desired final free Mg2ϩ
concentration, as calculated from the dissociation constants
K
d (ATP-Mg) ϭ 27.78 ␮M (54) and Kd (ADP-Mg) ϭ 240.96 ␮M (55)
at pH 8.0. Rca stock solutions purified in the presence of 0.5 mM
ADP were exchanged into buffers containing the appropriate
amount of ADP by using a PD-10 column (GE Healthcare),
such that dilution in the cuvette resulted in the desired final
ADP concentration during turnover measurements. For exam-
ple, 50 ␮l of Rca in buffer containing 1 mM ADP will result in 50
␮M ADP upon 20-fold dilution in the cuvette. In all kinetic
experiments presented here, the Rca concentration was kept
constant at 5.0 ␮M. Nominal final ADP concentrations of 0, 18,
42, 60, 112, and 132 ␮M were utilized, with each concentration
assayed in the presence of 0–4.0 mM ATP. The NADH assay
was employed to demonstrate that ultrapure ATP (Ն99%
Sigma or Alpha Aesar) contained 0.36–0.42% ADP contamina-
tion. Therefore, for the nominally zero-ADP reactions, the
ADP contamination resulted in about 4 ␮M ADP in reactions
containing 1 mM ATP. For each ADP concentration, the nor-
malized initial velocities were plotted as a function of substrate
concentration. Global curve fitting to Equation 2 was carried
out using the program OriginTM. The fitting procedure
involved fixing the Km at a constant value, fitting the Ki for ADP
inhibition globally, and fitting the kcat and nH values individu-
ally for each curve. Because the commercial ADP preparation
utilized in this study contains some amount of phosphate, the
maximum amount of ADP tolerated by the assay was shown to
be about 140 ␮M.
EnzCheck ATP Turnover Assays—To assay the ATPase activ-
ity of Rca, 700 ␮l of turnover working solution were added to
the sample and reference cuvettes; the A360 was autozeroed,
and 200 ␮l of MESG stock, 30 ␮l of PNP stock, 12 ␮l of 0.67 M
MgCl2 stock, and 50 ␮l of Rca (4.50 mg/ml solubilized in 25 mM
HEPES, pH 8.0, 300 mM KCl, 1 mM DTT, 10% glycerol) were
added to the sample cuvette, and the time scan was started.
After 60 s, 8 ␮l of ATP stock (500 mM) was added; the solution
was mixed using a manual cuvette mixer, and the A360 was
monitored for 300 s.
To determine the initial velocity of Rca turnover, the turn-
over time scans were corrected by subtracting the baseline col-
lected in the presence of ATP, and the slope of the steepest part
of the resulting curve (15–20 s) was determined (usually 30–50
s after addition of ATP). Care was taken to ensure that the
maximum absorbance value utilized to calculate the slope fell
within the linear range of the standard curve. The slope was
utilized to calculate the enzyme turnover velocity in terms of
micromolar phosphate produced per s by means of the standard
curve. This value was converted to turnover rates by normalizing
with respect to Rca subunit concentration (5.0 ␮M). Each reaction
was carried out in triplicate. The highest enzymatic turnover rate
that can be quantitatively measured with this assay was shown to
be equal to the production of 4.5 ␮M Pi/s.
nH
According to the protocol described above, the final Rca con-
centration in the cuvette was 5.0 ␮M; the ATP concentration
was 4.0 mM, and the total Mg2ϩ concentration was 9.00 mM.
Once coordination of one magnesium ion to ATP was taken
into account (Kd ϭ 27.78 ␮M at pH 8.6 (54)), the residual, i.e.
kcat ϫ ͓S͔
ko ϭ
(Eq. 2)
ADP ϫ Km
nH
K ϩ
m
ϩ ͓S͔
ͩ ͪ
Ki
EnzCheck Assays as a Function of Magnesium Concen-
free Mg2ϩ concentration, was calculated to be 5.0 mM. In solu- tration—Rca turnover data were determined at free Mg2ϩ con-
tion, coordination of a second magnesium ion to ATP occurs centrations of 1.0, 3.0, 4.0, 5.0, 6.5, 8.5, and 10 mM. As before,
with a Kd value of 10.2 mM (54). Therefore, a small fraction of free Mg2ϩ was calculated using published dissociation con-
the Mg2ϩ concentration termed “free Mg2ϩ” may be bound stants Kd (ATP-Mg) ϭ 27.78 ␮M (54) and Kd (ADP-Mg) ϭ 240.96 ␮M
OCTOBER 2, 2015•VOLUME 290•NUMBER 40
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Steady-state Turnover of Rubisco Activase
(55) at pH 8.0. For each free Mg2ϩ concentration, ATP concen-
Rubisco Reactivation Assays—The Rubisco reactivation
trations were varied between 0.05 and 4.0 mM, and the initial activity of different Rca preparations was measured using a
turnover rates were fit to Equation 1 to extract kcat and Km two-stage assay that employs coupling enzymes to determine
the concentration of 3-phosphoglycerate (3-PGA) produced
after 5 min of reactivation (56). Enolase from bakers’ yeast
and pyruvate kinase and lactic dehydrogenase from rabbit
muscle (PK-LDH) were purchased from Sigma. Di-phospho-
glycerate mutase preparations bearing a streptavidin tag
(dPGM-streptavidin) and tobacco Rubisco preparations
were a generous gift from Dr. Salvucci (United States
Department of Agriculture-ARS).
values. In these curve fits, a Hill coefficient nH ϭ 1.0 for ATP
hydrolysis provided the best fit to the data. The catalytic effi-
ciency kcat/Km was plotted against the free Mg2ϩ concentration
and fitted to Equation 3, where A represents baseline activity of
the ATP⅐Mg minimal substrate complex in the absence of free
Mg2ϩ; [Mg] represents the concentration of free magnesium
ions, and nH is the Hill coefficient.
For stage 1, stock solutions of tobacco Rubisco in the ECM
form were prepared in 100 mM Tricine-NaOH, pH 8.0, 10 mM
MgCl2, and 10 mM NaHCO3. Stock solutions of tobacco
Rubisco in the ER form were prepared in 20 mM Tricine-NaOH,
pH 8.0, 0.2 mM EDTA, 4 mM RuBP, and 5% PEG-3350. 1.25 ␮l of
Rubisco stock was added to 0.5-ml Eppendorf tubes containing
reaction buffer. The final volume of each reaction was 12.5 ␮l,
and the final reaction mixture contained 1.84 ␮M (1.0 mg/ml)
Rubisco holoenzyme, 5.0 ␮M (0.22 mg/ml) Rca, and 4.0 mM
ATP. For reactions with the ECM form, the final buffer solution
also contained 110 mM Tricine-NaOH, pH 8.0, 9.8 mM HEPES,
pH 8.0, 21.6 mM KCl, 2.07 mM DTT, 0.72% glycerol, 9.72 mM
MgCl2, 10.7 mM NaHCO3, 5 mM RuBP, and 5% PEG 3350. For
reactions with the ER form, the final buffer solution also con-
tained 102 mM Tricine-NaOH, pH 8.0, 9.8 mM HEPES, pH 8.0,
9 mM MgCl2, 10.7 mM NaHCO3, 21.6 mM KCl, 2.07 mM DTT,
0.72% glycerol, 0.02 mM EDTA, 5.4 mM RuBP, and 5.5% PEG-
3350. Reactivation mixtures were allowed to incubate for 5 min
at room temperature. The reactions were quenched by the
addition of 37.5 ␮l of hot (95 °C) 100 mM Tricine-NaOH, pH 8,
followed by 5 min of heating at 95 °C. Each reaction was vor-
texed briefly and centrifuged to remove the precipitate and
then stored at Ϫ20 °C.
kcat
nH
ϫ ͓Mg͔
ͩ ͪ
Km
kcat
Km
max
ϭ A ϩ
(Eq. 3)
nH
Kd ϩ ͓Mg͔
In control reactions, the ionic strength difference between 1.5
and 6.5 mM MgCl2 was accounted for by the addition of 15 mM
KCl to reactions containing 1.5 mM MgCl2. For these controls, a
full Michaelis-Menten curve was generated.
EnzCheck Assay in the Presence of Crowding Agents—The
effect of Ficoll as a crowding agent was tested using the stan-
dard cuvette assay described above. As described previously, a
standard curve working solution was prepared to a final volume
of 7.4 ml; however, 2.5 ml of 40% Ficoll 70 (w/v, Sigma) was
added while reducing the water aliquot to 3.754 ml. In this way,
the reaction mixture in the cuvette (1 ml) contained 10% Ficoll.
Similarly, the turnover working solution for the Rca activity
assays was prepared to a final volume of 7.00 ml as described
previously; however, part of the water aliquot was replaced with
2.5 ml of 40% Ficoll 70 to provide for a final Ficoll concentration
of 10% in the cuvette.
ATPase Assays in the Presence of Rubisco—Rubisco prepara-
tions isolated from tobacco leaves and flash-frozen in ammo-
nium sulfate were a generous gift from Michael Salvucci
(United States Department of Agriculture-ARS). On the day of
the experiment, an ammonium sulfate pellet was thawed on ice
and centrifuged for 10 min at 14,000 rpm, and the supernatant
was discarded. The pellet was resuspended in 170 ␮l of 20 mM
Tricine-NaOH, pH 8.0, and 0.2 mM EDTA buffer (inactivation
buffer). A Sephadex G-50 column was equilibrated in the same
buffer; excess buffer was removed, and Rubisco was loaded and
the column centrifuged for 2 min at 400 ϫ g. Rubisco prepara-
tions were subsequently concentrated using a centrifugal con-
centrator unit (Millipore, Amicon Ultracel 10K). Control reac-
tions containing Rubisco buffer blanks were carried out by
collecting the flow-through during the concentration step.
Desalted Rubisco in the E-form was either used directly for
experiments, or RuBP was added to 0.5 mM followed by a 2-h
incubation period (4 °C) to generate the E⅐RuBP form.
For stage 2, the 3-PGA content of each reaction was assayed
in 96-well UltraCruz௡ UV plates, utilizing a BioTek (Synergy
HT) plate reader. A four-enzyme assay mix was prepared con-
sisting of 100 mM Tricine-NaOH, pH 8.0, 10 mM MgCl2, 10 mM
NaHCO3, 20 mM KCl, 5 mM DTT, 0.5 mM NADH, 1.85 units of
pyruvate kinase, 2.33 units of lactic dehydrogenase, 0.5 units of
enolase, 0.5 units of diphosphoglycerate mutase, 0.2 mM 2,3-
diphosphoglycerate, and 2 mM ADP. For control reactions car-
ried out to ensure high activity of coupling enzymes, 177.8 ␮l of
the four-enzyme assay mix was placed into each well; the tem-
perature was equilibrated to 30 °C, and the initial A340 value was
recorded. 18.2 ␮l of water and 4 ␮l of 100 mM 3-PGA were
added, and the solution was mixed. After 7 min of incubation,
the final absorbance was recorded. To determine Rubisco
activity by measuring the total amount of 3-PGA produced
during the reactivation reaction, 177.8 ␮l of four-enzyme
assay mix were placed into each well, followed by the addi-
tion of 7.2 ␮l of H2O and equilibration at 30 °C. The initial
absorbance was recorded; 15 ␮l of quenched Rubisco re-ac-
tivation mix was added and incubated for 7 min, and the final
EnzCheck Microplate Assays—For reactions in the presence
of Rubisco, the EnzCheck assay was carried out in 200-␮l vol-
umes, using a Biotek Synergy HT microplate reader in combi-
nation with Corning NBS 96-well plates at 25 °C. The reduced
volume was necessary to perform experiments in the presence
absorbance was recorded. The difference in A340 was utilized
Ϫ1
of high Rubisco concentrations. The volumes of all solutions to calculate the amount of NADH oxidized (⑀340 ϭ 6220 M
were adjusted accordingly, and experiments were carried out cmϪ1), which is equal to the total amount of 3-PGA present
with and without 10% Ficoll.
in the sample.
24226 JOURNAL OF BIOLOGICAL CHEMISTRY
VOLUME 290•NUMBER 40•OCTOBER 2, 2015

Steady-state Turnover of Rubisco Activase
FIGURE 1. Tobacco Rca protein preparations and hydrolytic velocities. a, SDS-polyacrylamide gel documenting the purity of Rca expressed in E. coli and
affinity-purified. b, Rca turnover in the absence of ADP. Initial velocities were monitored using 5 ␮M Rca, 5 mM free Mg2ϩ, and 0–4 mM ATP by use of the
EnzCheck assay. Data were computer-fitted to Equation 1 using the program Origin, with kcat fitted individually for each data set, and Km and nH fitted globally
for all five data sets, which were derived from five different protein preparations.
Thermofluor Stability Assays—The Thermofluor assay was ␮M, which describes Mg2ϩ coordination to the ␤- and ␥-phos-
carried out essentially as described (35). Data were collected
using an Applied Biosystems ABI Prism 7900HT sequence
detection system. Stability experiments were performed using a
384-well polypropylene TempPlate PCR plate covered with
optically clear covers. Wells contained 20 ␮l of final volume
with tobacco ␤-Rca at 0.21 mg/ml (ϳ5 ␮M), 25 mM HEPES, pH
8.0, 250 or 228.7 mM KCl, and 300ϫ SYPRO Orange (prepared
from a 5000ϫ stock in DMSO with Nanopure water). Samples
with the lower KCl concentration were controls for the ionic
strength in the presence of MgCl2. The above samples were
prepared as two 1.1ϫ premixes (high and low KCl), from which
18-␮l aliquots were transferred into each well, followed by the
addition of 2 ␮l of concentrated stocks at variable concentra-
tions (e.g. ATP␥S, MgCl2, or both). Each individual condition
was measured in triplicate. Thermal denaturation experiments,
data collection, and analysis were performed as described pre-
viously (35).
phoryl groups of ATP (54).
The initial velocities as a function of ATP were computer-
fitted to a Michaelis-Menten-type steady-state turnover model
with inclusion of cooperativity (see Equation 1). Five data sets
generated from five different protein preparations provided inde-
pendent evidence of rate saturation near 1 mM ATP (Fig. 1b).
The extracted kcat values varied from one protein pool to
another, providing an average value of 22.3 Ϯ 4.9 turnovers
minϪ1 per subunit (22% relative population standard deviation)
for n ϭ 5 independent determinations (Table 1). The observed
range likely reflects the experimental errors associated with the
Bradford and EnzCheck assays. Typical differences in indepen-
dent Bradford concentration determinations were about 15%,
and for independent EnzCheck assays, between 6 and 25% dif-
ference in measured rates was observed.
Regardless, individual fitting of the five Michaelis-Menten
curves (Fig. 1b) provided essentially constant Michaelis con-
stants Km and Hill coefficients nH. Therefore, global curve fit-
ting procedures were applied to all five data sets using the pro-
gram Origin, with the parameters Km and nH restrained to
single values, while fitting kcat individually for each data set (Fig.
1b). Using this procedure, the Km value for ATP was extracted
to be 104 Ϯ 23 ␮M, and the Hill coefficient nH was extracted to
be 1.0 Ϯ 0.1 (population standard deviation determined by the
fitting algorithm) (Table 1). A Hill coefficient of unity implies a
lack of allosteric interactions, providing the surprising result
that ATP hydrolysis of oligomeric assemblies involves neither
positive nor negative cooperativity under these conditions (5.0
␮M apo-Rca with 5 mM free Mg2ϩ). Using the NADH assay (52),
no residual ADP was detected in apo-Rca preparations. How-
ever, trace amounts of ADP are introduced when adding ATP
to initiate turnover, as indicated by our measurements of 4 ␮M
ADP contamination in 1 mM ATP (28).
Results
ATPase Activity of Tobacco ␤-Rca Provides a Michaelis Con-
stant in the Mid-micromolar Range—To examine the regula-
tion of Rca turnover by the available ATP and ADP pools, a
continuous enzyme-coupled ATPase assay was developed that
monitors the kinetics of phosphate (Pi) release, while tolerating
moderate amounts of ADP in the reaction (EnzCheck assay)
(53). Near-homogeneous preparations of tobacco ␤-Rca were
produced by bacterial expression, affinity purification, and
enzymatic cleavage of the N-terminal His6 tag (Fig. 1a). To
generate apo-Rca, all purification procedures were carried out
in the absence of nucleotide. Each protein preparation yielded
at most 2 mg of Rca/liter of liquid culture, providing sufficient
material to measure the initial velocity of 5.0 ␮M Rca with ATP
concentrations ranging from 0 to 4 mM ATP (Fig. 1b). In this set
of experiments, the free Mg2ϩ concentration, i.e. Mg2ϩ not
coordinated to ATP, was kept constant at 5 mM. The concen-
To validate the results described above, Michaelis-Menten
tration of free Mg2ϩ was calculated using Kd (ATP-Mg) ϭ 27.78 curves were also generated using the NADH assay and then
OCTOBER 2, 2015•VOLUME 290•NUMBER 40
JOURNAL OF BIOLOGICAL CHEMISTRY 24227

Steady-state Turnover of Rubisco Activase
TABLE 1
ADP Provides Moderate Inhibition at Low ATP Concen-
trations—To investigate the regulation of Rca by the prevailing
ATP/ADP ratio, the ATPase assays were repeated in the pres-
ence of various amounts of ADP, again utilizing 5 ␮M tobacco
␤-Rca and 5 mM free Mg2ϩ. A series of Michaelis-Menten
curves were generated varying ATP from 0 to 4 mM, with each
curve generated in the presence of either 18, 42, 60, 90, 112, or
132 ␮M ADP in the reaction mixture (Fig. 2a). The maximum
amount of ADP tolerated by the assay was found to be about
140 ␮M, as higher concentrations lead to poor signal-to-noise
ratios due to residual Pi in commercial ADP preparations.
Curve fitting of individual data sets to Equation 1 suggested that
the apparent Km value (Km(app)) for ATP was increasing steadily
Catalytic parameters of tobacco apo-Rca derived from global curve
fitting procedures
Experiments were carried out at 5 ␮M Rca and 5 mM free Mg2ϩ. Data were fit to
Equation 1 (kcat fitted individually, and Km and nH fitted globally). Except for the
average kcat, indicated errors were estimated by the curve fitting algorithm.
Protein preparation
kcat
min
Km
mM
nH
Ϫ1
1
15.4 Ϯ 0.6
0.104 Ϯ 0.024
1.0 Ϯ 0.1
2
27.1 Ϯ 0.7
3
21.5 Ϯ 0.6
4
27.1 Ϯ 0.7
5
20.6 Ϯ 0.6
Average Ϯ S.D.
22.3 Ϯ 4.9 (n ϭ 5)
compared with the EnzCheck assay under identical conditions. with increasing ADP, in general agreement with competitive
In these experiments, tobacco ␤-Rca activity was monitored at product inhibition (Table 2). This effect became particularly
a subunit concentration of 5.5 ␮M, while varying the ATP con- apparent when normalizing each curve with respect to its kcat
centrations between 0.25 and 4.0 mM and keeping the total value, thereby illustrating the gradual shift of curves to higher
Mg2ϩ concentration constant at 5 mM. Using single data sets, [ATP] with increasing [ADP] (Fig. 2b). As determined previ-
extracted kcat values were 20.2 Ϯ 1.1 minϪ1 for the EnzCheck ously, turnover numbers of different protein preparations var-
assay and 15.3 Ϯ 0.8 minϪ1 for the NADH assay, whereas the ied substantially. Although a weak trend in kcat could be dis-
extracted Km values were 136 Ϯ 50 and 122 Ϯ 45 ␮M, respec- cerned in the presence of ADP, the average kcat value (20.1 Ϯ 6.8
tively (Equation 1). The standard deviations provided here were minϪ1, n ϭ 6, Table 2) cannot be distinguished from that deter-
estimated by the curve fitting algorithm. Because a better esti- mined in the absence of ADP (22.3 Ϯ 4.9 minϪ1, n ϭ 5, Table 1).
mate of the true error is obtained by fitting multiple data sets as To examine whether kcat changes as a function of ADP, Rca
described above (Fig. 1b), it appears that the kinetic constants hydrolytic activity was measured with increasing ADP while
extracted from the two assays fall within experimental error of keeping ATP and free Mg2ϩ constant (2 and 5 mM, respec-
each other.
tively). The data were normalized with respect to the turnover
To examine whether the variations in hydrolytic kcat values rate observed at zero ADP, and the experiment was repeated
observed for different protein preparations (Fig. 1b) result in three times with independently prepared protein preparations
different Rubisco reactivation activities, three different Rca (Fig. 2c). The results clearly demonstrate that hydrolytic activ-
preparations were tested. At 4 mM ATP, 5 mM free magnesium, ity is not activated by micromolar amounts of ADP. The mea-
and 5 ␮M Rca, the hydrolytic turnover numbers were deter- sured percent hydrolytic activity remaining with increasing
mined to be 27.5 Ϯ 1.3, 19.7 Ϯ 1.2, and 25.4 Ϯ 2.2 minϪ1. On the ADP (Fig. 2c) follows closely the activity calculated using the
same day, 5 ␮M Rca from each preparation was incubated for 5 kinetic constants extracted from our competitive inhibition
min with tobacco Rubisco in either the ER or the ECM form. model (see below and Table 3). Based on these data, we con-
Upon heat denaturation, the total amount of 3-PGA produced clude that within the range tested, kcat does not depend on ADP
was measured spectrophotometrically using the coupling concentrations.
enzymes enolase, diphosphoglycerate mutase, pyruvate kinase,
The ADP Inhibition Constant Is Estimated to be One-third of
and lactic dehydrogenase (56). For each Rca preparation, per- the Km Value for ATP Hydrolysis—To extract precise kinetic
cent Rca reactivation activity was calculated by comparing the constants, the five ADP-dependent Michaelis-Menten curves
amount of 3-PGA produced in reactions containing the ER (Fig. 2a) were subjected to global curve fitting procedures, uti-
form with that produced in reactions containing the ECM form. lizing a steady-state turnover model with inclusion of compet-
Overall, Rubisco reactivation activities appeared to follow the itive inhibition (Equation 2). Using the program Origin, the Km
same trend as hydrolytic activities, with relative reactivation value was fixed to 104 ␮M (Table 1), and the ADP inhibition
activities determined to be 88.4 Ϯ 26.4, 72.5 Ϯ 11.8, and 87.5 Ϯ constant Ki was fitted as a global parameter. At the same time,
18.7% for the three Rca preparations. However, the similarity of the kcat and nH values were fitted individually for each data set
values and associated errors do not allow for a quantitative dis- (Fig. 2a). In this way, the Ki value for ADP was extracted to be
tinction of measured reactivation activities.
37 Ϯ 7.0 ␮M (Table 3), about one-third of the Km value for ATP,
Surprisingly, cotton ␤-Rca was shown to turn over five to six raising the possibility of tighter product binding compared with
times more slowly than tobacco ␤-Rca, with an estimated turn- substrate and reflecting previously reported thermal stability
over rate of 3.8 Ϯ 1.2 minϪ1 (n ϭ 6) under substrate-saturation data (28).
conditions (28). In these preliminary experiments, we have esti-
ADP Is Essential for Positive Subunit Cooperativity—Surpris-
mated a Km value for ATP hydrolysis of about 1.5 mM, suggest- ingly, the individual Michaelis-Menten curves adopted a dis-
ing substantially weaker substrate binding compared with tinctly sigmoidal shape with rising ADP concentrations (Fig.
tobacco ␤-Rca. Because the low hydrolytic rate of cotton ␤-Rca 2b), and curve fitting provided a consistently increasing Hill
observed in our laboratory yielded relatively weak and noisy coefficient (Table 3). At zero ADP, nH was best fit with a value of
signals, mechanistic work on this variant was not pursued 1.0 Ϯ 0.1 (Table 1), whereas at 132 ␮M ADP, the best-fitted nH
further.
value was extracted to be 1.9 Ϯ 0.2 (Table 3). These values
24228 JOURNAL OF BIOLOGICAL CHEMISTRY
VOLUME 290•NUMBER 40•OCTOBER 2, 2015

Steady-state Turnover of Rubisco Activase
FIGURE 2. Effect of ADP on Rca turnover. a, ADP ranging in concentration from 18 to 132 ␮M was added to tobacco Rca preparations prior to initial rate
measurements as a function of ATP concentration. Curve fits to Equation 2 are shown. The inhibition constant Ki for ADP was fitted globally, whereas kcat and
nH were fitted individually for each data set (Table 3). The Km value was fixed to 104 ␮M (Table 1). (Each data set was also fitted individually to Equation 1, see
Table 2.) b, data from a replotted upon normalization with respect to kcat values. c, initial turnover velocities measured as a function of ADP concentration and
normalized with respect to 0 ADP. Experiments were carried out at 2 mM ATP and 5 mM free magnesium ion concentration and utilizing three different protein
preparations. At each ADP concentration, the average percent rate and its standard deviation were calculated for 3–6 individual rate determinations (red data
points). For each ADP concentration, the expected percent activity remaining was calculated from the kinetic constants shown in Table 3 (blue data points).
TABLE 2
TABLE 3
Catalytic parameters of ADP-bound tobacco Rca derived from individ- Catalytic parameters of ADP-bound tobacco Rca derived from global
ual curve fitting procedures
curve fitting procedures
Experiments were carried out at 5 ␮M Rca and 5 mM free Mg2ϩ. The data sets were
fit individually to Equation 1 for each ADP concentration, and the apparent catalytic
efficiency kcat/Km (app) was calculated from the fitted parameters. Indicated errors
were estimated by the curve fitting algorithm and propagated to estimate the error
on the apparent catalytic efficiency.
Experiments were carried out at 5 ␮M Rca and 5 mM free Mg2ϩ. The data sets were
fit to Equation 2 with a fixed Km value of 0.104 mM. The Ki value for ADP was fit
globally, whereas kcat and nH values were fit individually for each ADP concentra-
tion. Indicated errors were estimated by the curve fitting algorithm.
͓ADP͔
Km
Ki
mM
kcat
min
nH
͓ADP͔
nH
kcat
min
Km (app)
mM
0.152 Ϯ 0.034 84.7 Ϯ 19.3
0.272 Ϯ 0.033 52.6 Ϯ 6.5
0.274 Ϯ 0.049 68.9 Ϯ 12.6
0.440 Ϯ 0.048 56.0 Ϯ 6.2
0.359 Ϯ 0.059 86.8 Ϯ 14.5
0.428 Ϯ 0.067 43.4 Ϯ 6.8
k
cat/Km (app)
Ϫ1
␮M
mM
Ϫ1
Ϫ1
␮M
minϪ1 mM
18
0.104 mM
0.037 Ϯ 0.007
12.9 Ϯ 0.5
14.0 Ϯ 0.5
18.8 Ϯ 0.6
23.9 Ϯ 0.6
31.7 Ϯ 0.8
18.7 Ϯ 0.8
1.2 Ϯ 0.1
1.5 Ϯ 0.1
1.6 Ϯ 0.1
1.8 Ϯ 0.2
1.8 Ϯ 0.1
1.9 Ϯ 0.2
18
1.2 Ϯ 0.1 12.9 Ϯ 0.6
1.5 Ϯ 0.1 14.3 Ϯ 0.3
1.6 Ϯ 0.1 18.9 Ϯ 0.7
1.8 Ϯ 0.2 24.6 Ϯ 0.5
1.8 Ϯ 0.1 31.2 Ϯ 1.0
1.9 Ϯ 0.2 18.6 Ϯ 0.6
42
42
60
60
90
90
112
132
112
132
Average Ϯ S.D.
20.1 Ϯ 6.8 (n ϭ 6)
ADP may occupy additional sites within Rca oligomers, thereby
indicate that at least two subunits interact with each other, causing weak inhibition of ATP binding. At the same time,
providing for a net positively cooperative system. The highest these observations provide evidence that in oligomeric Rca
ADP concentration accessible by this assay, 132 ␮M, equals 3–4 assemblies, a fraction of subunits must be occupied by ADP to
times the Ki value for ADP binding, suggesting that at this point achieve an allosterically regulated system.
OCTOBER 2, 2015•VOLUME 290•NUMBER 40
JOURNAL OF BIOLOGICAL CHEMISTRY 24229

Steady-state Turnover of Rubisco Activase
FIGURE3. EffectoffreeMg2؉ onthecatalyticefficiencyofATPhydrolysisbytobaccoRca. a, initialratesweremonitoredasafunctionofATPconcentration
while varying the free magnesium ion concentration from 0.10 to 10 mM (Table 4). For clarity, four out of 10 curves are shown, each normalized with respect to
the kcat value. Each data point represents the average of three measurements. The data were fit to Equation 1 with nH ϭ 1. For each free [Mg2ϩ], kcat and
apparent Km values were extracted from the curve fits, and the catalytic efficiency kcat/Km(app) was calculated. b, catalytic efficiency as a function of free [Mg2ϩ
]
was fit to Equation 3, and the Kd and nH values for binding of the second magnesium ion were extracted. Inset, thermostability of tobacco Rca at low (0–1 mM)
free [Mg2ϩ] and high (3–5 mM) free [Mg2ϩ], as measured by the Thermofluor assay.
Magnesium-mediated Activation of Rca Hydrolytic Activity—
TABLE 4
Catalytic parameters of tobacco apo-Rca as a function of free mag-
Although substantial magnesium-dependent enhancement of
nesium ion concentration, derived from individual curve fitting
Rca hydrolytic activity was reported some years ago (57, 58), a
procedures
detailed mechanistic description of this phenomenon has not
Experiments were carried out at 5 ␮M Rca. The data sets were fit individually to
Equation 1 for each ADP concentration, and the catalytic efficiency kcat/Km (app) was
yet been provided. In solution, ATP is known to tightly coordi-
calculated from the fitted parameters. In all cases, the best fits were obtained with a
nate one magnesium ion, a feature that appears critical for Rca-
Hill coefficient nH ϭ 1.0. Indicated errors are estimates provided by the curve fitting
algorithm. NA, not applicable.
mediated hydrolysis. Our data indicate that the complete
͓Mg2؉
͔
kcat
min
Km (app)
mM
k
cat/Km (app)
absence of magnesium ions abolishes enzymatic turnover.
Moreover, noncoordinated ATP appears to compete with
ATP⅐Mg for binding to the active site, as the addition of 3 mM
free ATP to 5 mM ATP⅐Mg results in decreased turnover rates.
We conclude that the minimal Rca substrate consists of
ATP⅐Mg. However, Rca hydrolytic activity increases substan-
tially upon addition of Mg2ϩ in excess of that tightly coordi-
nated to ATP (58), suggesting that a second protein-based mag-
nesium-binding site may serve as co-activation site (57).
free
Ϫ1
Ϫ1
mM
minϪ1 mM
0.1
1.0
1.5
2.0
3.0
4.0
5.0
6.5
8.5
10.0
16.4 Ϯ 1.1
23.5 Ϯ 0.8
27.6 Ϯ 1.1
21.3 Ϯ 0.8
24.1 Ϯ 0.5
20.9 Ϯ 0.5
22.8 Ϯ 0.6
19.1 Ϯ 0.6
20.7 Ϯ 0.5
20.7 Ϯ 0.3
0.453 Ϯ 0.110
0.323 Ϯ 0.046
0.292 Ϯ 0.051
0.119 Ϯ 0.021
0.113 Ϯ 0.014
0.079 Ϯ 0.014
0.083 Ϯ 0.021
0.069 Ϯ 0.014
0.067 Ϯ 0.011
0.068 Ϯ 0.005
NA
36.3 Ϯ 9.2
72.8 Ϯ 10.6
94.4 Ϯ 16.6
179 Ϯ 32
214 Ϯ 27
264 Ϯ 47
275 Ϯ 70
277 Ϯ 57
309 Ϯ 51
304 Ϯ 23
NA
Average Ϯ S.D.
21.7 Ϯ 3.0 (n ϭ 10)
To examine activation by binding of a second Mg2ϩ ion, the
ATP-dependent turnover rate of tobacco apo-Rca was deter-
mined as a function of free Mg2ϩ between 0.1 and 10 mM. A
total of 10 Michaelis-Menten curves were generated by varying
ATP between 0 and 4 mM for each curve, while keeping free
Mg2ϩ at a constant value and measuring each data point in
triplicate (Fig. 3a). Each curve was fit to Equation 1 to extract
hydrolyzed more rapidly, when a total of two magnesium ions
are coordinated to the Michaelis complex.
For each curve, the catalytic efficiency kcat/Km(app) was plot-
ted as a function of the free Mg2ϩ concentration (Fig. 3b). Based
on the observed saturation behavior, these data were fit to a
steady-state model describing augmentation of base level activ-
ity upon binding of a second Mg2ϩ ion (Equation 3). Because of
the distinctly sigmoidal shape of this curve, a Hill coefficient for
magnesium-mediated activation (nH-Mg) was included in the
model. By curve fitting, the apparent Kd value for the second
Mg2ϩ-binding site was estimated to be 6.8 Ϯ 2.6 mM. Based on
the extracted Hill coefficient nH-Mg ϭ 2.5 Ϯ 0.5, Mg2ϩ binding
is judged to be a cooperative process that involves at least three
Rca subunits. The catalytic efficiency is shown to increase 8.4-
fold with rising free magnesium, a substantial elevation from a
k
cat and Km(app) values (Table 4). In all cases, a lack of coopera-
tivity (nH ϭ 1) provided the best fit to the data, in full agreement
with our observations described above in the absence of ADP.
An examination of extracted kcat values did not suggest a Mg2ϩ
-
related trend (Table 4) and provided an average kcat of 21.7 Ϯ
3.0 minϪ1 (n ϭ 10), within error identical to the average values
reported in Tables 1 and 2.
In contrast, the extracted Km(app) values followed a steady
downward trend with increasing Mg2ϩ ions, with magnitudes
decreasing from 453 Ϯ 110 ␮M at 0.1 mM free Mg2ϩ to 69 Ϯ 14
␮M at 6.5 mM free magnesium (Table 4). These data provide baseline value of 36.3 Ϯ 9.2 minϪ1/mM for ATP⅐Mg to a maxi-
evidence that ATP binds more tightly to the Rca active site, or is mum of 309 Ϯ 51 minϪ1/mM for ATP⅐Mg⅐Mg (Table 4).
24230 JOURNAL OF BIOLOGICAL CHEMISTRY
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Steady-state Turnover of Rubisco Activase
FIGURE 4. Simulations of predicted normalized velocities as a function of adenine nucleotide concentration. Calculations were carried out using
Equation 2, while fixing the Hill coefficient nH for ATP hydrolysis at either 1, 2, or 4, as indicated in a–c, respectively. The following parameters were utilized to
calculate initial velocities (Table 3): kcat ϭ 22.3 minϪ1, Km ϭ 104 ␮M, and Ki ϭ 37 ␮M, values that were extracted in the presence of saturating magnesium (free
[Mg2ϩ] ϭ 5 mM). All simulations were carried out keeping the available adenine nucleotide concentration (ATP ϩ ADP) constant at either 0.1, 0.2, 0.5, 1, 2, or 5
mM. For each nucleotide concentration, Rca activity was calculated as a function of the mole fraction of adenosine triphosphate, i.e. [ATP]/([ATP ϩ ADP]).
Because of the allosteric behavior, the steepest increase is nucleotide pool, the extracted kinetic constants were utilized to
observed between 1 and 2 mM free Mg2ϩ with half-maximal predict turnover velocities at nucleotide concentrations rang-
efficiency at ϳ1.5 mM free Mg2ϩ (Fig. 3b).
ing from 0.1 to 5 mM (Equation 2). The Hill coefficient for ATP
Control reactions carried out by adjusting the ionic strength turnover was fixed at either 1.0, 2.0, or 4.0 (Fig. 4, a–c, respec-
with KCl rather than with MgCl2 provided clear evidence that tively), and normalized velocities V0/Vmax were calculated
the increased catalytic efficiency at high [Mg2ϩ] is due to an using the constants Km ϭ 104 ␮M and Ki ϭ 37 ␮M (Table 3),
increase in divalent cation, rather than an increase in ionic values determined in the presence of saturating magnesium
strength. Similarly, control reactions were carried out in the (free [Mg2ϩ] ϭ 5 mM). As expected, activities increase with
presence of MnCl2 rather than MgCl2. In these reactions, 5 ␮M rising amounts of available nucleotide until saturation is
Rca and 0.5 mM ATP were combined with 5.5 mM total divalent observed. Because of the moderate degree of product inhibition
cation. In the presence of Mg2ϩ, the turnover rate was mea- (Km Ϸ3Ki), slightly convex velocity curves are obtained in the
sured to be 8.3 Ϯ 0.3 minϪ1, whereas in the presence of Mn2ϩ
,
absence of cooperative behavior (nH ϭ 1) (Fig. 4a). The intro-
the rate was measured to be only 0.26 Ϯ 0.02 minϪ1. The duction of cooperativity (nH ϭ 2 or 4) produces a sigmoidal
32-fold drop in activity when replacing magnesium with man- shape that becomes more pronounced at higher nH values (Fig.
ganese ions clearly indicates that catalytic activation is specific 4, b and c). Specifically, the simulated curves predict that with
to magnesium coordination, rather than the result of a more nH ϭ 2, Rca will operate between 10 and 80% capacity, if the
general divalent cation effect.
available nucleotide pool equals 1 mM (Fig. 4b, blue curve).
Thermofluor assays indicate an ϳ2 °C increase in the appar- Under these conditions, about 70% of maximal ATPase activity
ent mid-point for protein denaturation (Tm (app)), when free would be achieved at high energy charge (ATP/ADP ϭ 9:1) but
[Mg2ϩ] is raised from 1.0 to 3.0 mM (Fig. 3b, inset). Therefore, only about 15% activity at low energy charge (ATP/ADP ϭ 1:1).
magnesium-mediated activation appears to be accompanied by This example demonstrates the feasibility of substantial Rca
thermal stabilization of Rca assemblies, a feature that is consis- down-regulation, if ADP were to accumulate in the stroma.
tent with the formation of topologically closed rings.
Neither Crowding Agents nor Elevated Concentrations of
In combination, the observed positive subunit cooperativity Rubisco Modulate Rca Hydrolytic Activity—To evaluate
and the reduced Km value suggest that subunit-subunit interac- excluded volume effects due to macromolecular crowding, the
tions are mediated by a magnesium ion located in or near the ATPase activity of tobacco ␤-Rca was measured in the presence
active site bearing the minimal substrate ATP⅐Mg. Therefore, of 10% Ficoll-70, an inert carbohydrate polymer (59). At this
the second metal ion may not only coordinate ATP but also concentration, the experimental volume fraction of Ficoll-70
bridge molecular contacts between adjacent protein chains. has been estimated to be about 7%, a condition that was shown
This idea is further supported by previously published results to provide a 5-fold increase in the enzymatic activity of phos-
from our laboratory on magnesium-mediated assembly of cot- phoglycerokinase (59). Therefore, the ATPase activity of
ton ␤-Rca (28). We suggest that the fully activated Michaelis tobacco ␤-Rca was analyzed with and without Ficoll using the
complex may be best represented by Rca⅐Mg⅐ATP⅐Mg, and may EnzCheck assay. Kinetic data were collected in triplicate on 5
form upon light adaptation in the stroma.
␮M Rca, 4 mM ATP, and 5 mM free Mg2ϩ. Surprisingly, we found
Predicted Turnover Rates Provide Evidence for Incremental that Ficoll-70 had no discernible effect on Rca hydrolytic activ-
Regulation by the Prevalent ATP/ADP Ratio—In general, the ity. The average turnover rates Ϯ Ficoll exhibited standard
regulation of Rca oligomeric assemblies may be afforded by a deviations of 11.4 and 5.0%, and differed from each other by
combination of product inhibition and subunit cooperativity. only 12.4%, well within the estimated error of ϳ22% for kinetic
To examine Rca regulation by the available (i.e. unbound) experiments (Table 1).
OCTOBER 2, 2015•VOLUME 290•NUMBER 40
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Steady-state Turnover of Rubisco Activase
To examine the effect of high Rubisco concentrations on Rca reactivation activities of cotton ␣- and ␤-Rca has demonstrated
hydrolytic activity, the ATPase assays were repeated in a micro- that ATPase activity follows the same trend as Rubisco reacti-
plate reader to reduce the reaction volume. Inactivated Rubisco vation activity (46). The authors reported that compared with
isolated from tobacco leaves was added either in its E- or ER- cotton ␣-Rca, 2 ␮M cotton ␤-Rca exhibited substantially less
form. The E-form was prepared by incubation with EDTA to ATPase and activase activity, whereas mixing of the two iso-
decarbamylate active sites, and the ER-form was generated by forms augmented turnover rates.
subsequent addition of 500 ␮M RuBP. ATPase assays were per-
Tobacco ␤-Rca Assembly States—Because of its high activity,
formed with 5 ␮M Rca and Rubisco concentrations ranging the mechanistic studies reported here were carried out on
from 1.4 to 20 ␮M. At the highest Rubisco concentrations tobacco ␤-Rca at 5 ␮M subunit concentration, either in the apo-
tested, the molar ratio of Rubisco holoenzyme to Rca hexamer form (Fig. 1) or the ADP-bound form (Fig. 2), with ATP added
was 24 (Rubsico/Rca6 ϭ 24:1). Even under these conditions, the to initiate hydrolytic turnover. Tobacco Rca is known to form
observed turnover rates remained essentially identical to the heterogeneous mixtures under most conditions. However,
rates observed in the absence of Rubisco. To promote more the weight-averaged sedimentation coefficient was recently
highly associated states, the effect of 10% Ficoll-70 was tested in reported to be consistent with a hexameric state at concentra-
samples containing 20 ␮M Rubisco. However, as determined tions of ϳ0.2 mg/ml (ϳ5 ␮M) or higher, without any discern-
previously, no difference in hydrolytic rates could be discerned, able differences as a function of nucleotide status (29). These
as the average hydrolytic rates (n ϭ 3) measured with and with- data strongly suggest that hexamers are present in the reaction
out Ficoll differed by only 1%.
mixtures studied here, as all kinetic assays were carried out at 5
␮M tobacco Rca. Although average particle sizes as a function of
Rca concentration have been published (29), the microscopic
assembly equilibria of tobacco Rca have not yet been deter-
mined. Therefore, the percent hexamer remains unknown, and
our kinetic analysis of tobacco Rca can only be interpreted in
terms of average turnover rates per subunit. In contrast to
tobacco ␤-Rca, no more than 50% of cotton ␤-Rca appears to
form hexamers at 5 ␮M concentration (28), underscoring the
substantial species-specific differences not only in activity but
also in assembly.
Discussion
Hydrolytic Activity Appears to Vary Greatly among Higher
Plant Rca Proteins—We have developed a continuous enzyme-
linked ATPase assay based on the production of phosphate (53).
This assay has opened the door to Rca enzyme kinetic studies
that interrogate the effect of ADP in the reaction mixture. Tra-
ditionally, Rca ATPase activity has been measured by monitor-
ing the production of ADP (52), an approach that limits mech-
anistic studies, because the ATP/ADP ratio cannot be adjusted
in the reaction mixture. Utilizing both assay types, we have
determined tobacco ␤-Rca and cotton ␤-Rca hydrolytic activi-
ties as a function of substrate concentration. We found that the
kcat value of tobacco Rca determined in this work, 22.3 Ϯ 4.9
minϪ1 (Table 1), is substantially lower than previously reported
values, which range from 45 to 60 minϪ1 (32, 45). In this work,
Rca hydrolytic activity was determined at a subunit concentra-
tion of 5 ␮M, and in published works, the activity was measured
utilizing 2 ␮M Rca or less. A spike in ATPase activity has been
reported to occur at 1–2 ␮M subunit concentration, followed by
a downward trend at somewhat higher concentrations (30).
In support of this notion, preliminary data in our laboratory
indicate a substantial reduction in turnover rate with
increasing protein concentration.3 This effect, likely related
to concentration-dependent assembly equilibria (29), fully
accounts for the discrepancy observed between our turnover
rates and those reported previously. Similarly, in our hands,
cotton ␤-Rca turns over about 4-fold more slowly than pre-
viously reported (46), again suggesting that assembly equi-
libria may modulate hydrolytic activity.
Previous reports have shown that the minimal oligomeric
unit for ATP hydrolysis consists of about three subunits (30,
32). It is not surprising that the estimated stoichiometry for
ATP hydrolysis is smaller than that for Rubisco reactivation,
ϩ
because in AAA proteins, active sites functional in hydrolysis
are formed at the molecular interface of two subunits. However,
Rubisco reactivation must couple ATP hydrolysis to mechani-
cal work, a process that likely requires six-subunit rings to form
the reactivation-competent state (32), lending biological rele-
vance to the mechanistic interpretations presented here.
Allosteric Regulation Is a Function of ADP Binding—Using
the steady-state approximation, we have obtained Hill coeffi-
cient nH ϭ 1.0 for ATP hydrolysis in the absence of ADP and
nH ϭ 1.9 in the presence of 132 ␮M ADP (Tables 1 and 2). These
values suggest that an ATP-bound Rca subunit cannot allosteri-
cally interact with a neighboring ATP-bound or empty subunit.
Instead, such an interaction appears to require a partner sub-
unit occupied by ADP, indicating that positive cooperativity
involves at least two sites. We conclude that the presence of
ADP is absolutely critical in facilitating functional communica-
tion between subunits (Fig. 2, a and b). However, we do not
observe any activation of hydrolytic rate at micromolar ADP
concentrations (Fig. 2c), although such an effect was recently
reported for the distant homolog ClpX (60). In ClpX, ϳ100 ␮M
ADP is thought to set the homo-oligomeric ring into a catalyt-
ically activated conformational state, as evidenced by a moder-
ate jump in ATPase activity.
Notably, we found that tobacco operates with a 5–6-fold
higher kcat value than cotton ␤-Rca. The existence of substan-
tial activity variations among higher plant Rca proteins has
been noted previously, although the physiological significance
of these species-specific differences remains poorly understood
(10). However, ␤-Rca is the only isoform expressed in tobacco,
whereas cotton expresses both ␣- and ␤-isoforms of Rca. Some
years ago, a direct comparison of ATP hydrolytic and Rubisco
The larger Km value for ATP (104 ␮M) compared with the Ki
value for ADP (37 ␮M) may imply that positive subunit cooper-
ativity is a result of stronger ADP interaction with the active site
3 S. Hazra and R. M. Wachter, unpublished results.
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Steady-state Turnover of Rubisco Activase
compared with ATP. Although the Kd for ATP remains
unknown, tighter ADP binding has been suggested based on
relative thermostabilities (28). The relative Km and Ki values
(Ki/Km ϭ 0.36) suggest that the availability of excess ATP over
ADP may be critical in accessing conformational states that
promote hexameric ring closure (see below).
Light-dependent Magnesium Activation—In this work, we
demonstrate that the apparent Km value for ATP hydrolysis is
reduced ϳ6.6-fold in the presence of high Mg2ϩ concentra-
tions (Table 4). This effect leads to a substantial regulation of
ATP turnover rates as a function of available (unbound) mag-
nesium ions, with half-maximal catalytic efficiency observed at
about 2 mM free [Mg2ϩ]. In previous works, we and others have
demonstrated that excess magnesium ions increase the binding
affinity of ATP (28, 35, 61). Recently, we have reported that
magnesium concentrations in the low millimolar range pro-
mote cotton ␤-Rca assembly to hexameric states and disfavor
the formation of larger supramolecular complexes (28). In line
with this observation, we propose that cation binding involves
both coordination to ATP in the active site of one protomer and
coordination to several acidic residues located on the adjacent
protomer (28). Although the Mg2ϩ dependence of tobacco Rca
assembly has not yet been investigated, we demonstrate here
that 5 ␮M tobacco Rca is stabilized by ϳ2 °C when free [Mg2ϩ
]
is raised above 2 mM (Fig. 3b, inset). An increase in thermal
stability would be consistent with the formation of topologi-
cally closed toroidal rings from open hexameric assemblies. In
combination, it appears plausible that the structural basis of
Mg2ϩ activation consists of ring closure of six-subunit assem-
blies, whereas open ring-like hexamers may provide a template
for supramolecular assembly (Fig. 5) (28).
Magnesium ion concentrations are known to vary in the
stroma as a function of proton pumping across the thylakoid
membranes. In dark-kept intact chloroplasts, available [Mg2ϩ
]
has been estimated to be 0.50 Ϯ 0.53 mM, whereas a 10-min
illumination period has provided an estimate of 1.95 Ϯ 0.97 mM
FIGURE5. Hypotheticalmodelforsiteasymmetryandmagnesiumactiva-
tion in hexameric Rca assemblies. Based on the Hill coefficients presented
in this work, hexameric Rca assemblies are proposed to operate with positive
cooperativity of at least three protomers. One Rca6 toroid may consist of sites
occupied by ATP, of sites occupied by ADP, and of empty sites. The minimal
substrate for hydrolysis is ATP⅐Mg, whereas the second Mg2ϩ ion serves as
co-activator by mediating proper subunit-subunit contacts. The proposed
mechanistic model involves an open ring-like assembly at low magnesium
ion concentrations. When Mg2ϩ rises upon light exposure of the chloroplast,
bindingoftheco-activatorMg2ϩ atasecondsitemayleadtotheformationof
topologically closed hexameric toroids, with concomitant increase in cata-
lytic efficiency by about 1 order of magnitude.
free [Mg2ϩ] (62). As the pH of the stroma rises, [Mg2ϩ
]
increases to provide electrostatic compensation, thereby up-
regulating Rca hydrolytic activity to reactivate Rubisco for CO2
fixation. This notion would provide a plausible answer to an as
yet unresolved question of how uncontrolled Rca-mediated
ATP hydrolysis is prevented in the stroma.
Different Classes of Sites May Promote Functionally Relevant
Ring Asymmetry—In a toroidal assembly, communication
across subunits may require coordinated conformational teins form monodisperse hexamers for which sub-stoichiomet-
changes within the ring that exhibit substantial energy barriers. ric ATP binding has been demonstrated (50, 63). In addition to
The low degree of subunit cooperativity observed in this work empty sites, high and low affinity nucleotide-binding sites have
(nH Ϸ2) supports pairwise binding of ATP, implying that three been characterized, suggesting a sequential arrangement of
different classes of sites may co-exist in ring-like assemblies tight-weak-empty-tight-weak-empty sites in hexameric toroids
(Fig. 5). Substantial support for the existence of three classes of (64). Extracted Hill coefficients (nH) indicate positive coopera-
sites is provided by the Hill coefficient for magnesium activa- tivity between 2 or 3 sites, in support of a stochastic or coordi-
tion of hydrolytic activity (nH (Mg) ϭ 2.5). This coefficient nated cyclic pattern of hydrolysis (63, 64). The “symmetry mis-
ϩ
implies that at least three different classes of sites cooperate to match” problem in this system (six AAA domains stacked
achieve a high catalytic rate upon binding of a second Mg2ϩ ion. onto the seven-subunit proteasome outer ring) has led to the
According to this model, the nucleotide occupation of sites may idea that at any one time only two subunits associate with the
be coordinated with empty sites within the ring, in line with partner protein. Similarly, the stoichiometric mismatch
ϩ
previously proposed models for ClpX and PAN, distant rela- described for the six-subunit AAA rings formed by N-ethyl-
ϩ
tives of Rca among the classic clade AAA proteins. These pro- maleimide-sensitive factor interacting with its 4-fold symmet-
OCTOBER 2, 2015•VOLUME 290•NUMBER 40
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Steady-state Turnover of Rubisco Activase
ric partner protein has recently been attributed to asymmetric
interaction geometries during the catalytic cycle (65).
The hinges forming the nucleotide binding pockets and con-
Loading of Rca Assemblies with the Rubisco Substrate Does
Not Modulate Turnover—In general, the ATP turnover rate of
ϩ
AAA proteins may be stimulated, dampened, or unaffected by
ϩ
the partner protein. Although literature reports have suggested
that Rubisco does not affect ATP turnover, the Rubisco concen-
trations used in these assays were significantly lower than those
of Rca, e.g. ϳ1.5 ␮M Rubisco holoenzyme in combination with
4.7 ␮M Rca (32). Therefore, we have tested the effect of high
Rubisco concentrations, up to 24 Rubisco holoenzymes per Rca
hexamer, under conditions where ϳ7% of the volume is occu-
pied by a macromolecular carbohydrate-based crowding agent.
In support of previous results, these conditions did not signifi-
cantly alter Rca hydrolytic rates, confirming that the partner
protein is not involved in the regulation of Rca ATPase activity.
Under in vivo conditions, where excluded volume effects are
estimated to be about 50%, Rca may not be able to fully engage
with Rubisco until assemblies are activated by a rise in stromal
magnesium in response to light.
necting the AAA N- and C-domains are thought to adjust
continuously during catalysis (64, 66). The general picture
emerging from these studies suggests that the population of
ϩ
AAA conformational states is tightly coupled to the types of
nucleotides bound in the active sites.
Predicted Catalytic Activities under Varying Environmental
Conditions—In the chloroplast stroma, Rca activity is thought
to be substantially modulated by the mole fraction of adenosine
triphosphate, [ATP]/([ATP ϩ ADP]), with the total pool of ade-
nine nucleotides remaining constant at about 5 mM (10). The
hydrolytic activities calculated using the constants determined
here demonstrate that ATPase activity strongly depends on the
adenine nucleotide pool (Fig. 4). Unfortunately, reliable esti-
mates of unbound adenine nucleotide concentrations in the
stroma are not readily available. Therefore, the expected levels
of in vivo Rca hydrolytic activity remain difficult to delineate.
Our calculations suggest that at high magnesium a free nucle-
otide pool of 1 mM may provide for appropriate biological reg-
ulation as a function of the available ATP/ADP ratio, covering a
range from 90% under all-ATP conditions to 15% at low ATP
(ATP/ADP ϭ 1). Regardless, none of the predicted curves are
steeply convex in appearance, indicating that tobacco ␤-Rca
does not operate in a switch-like fashion at high magnesium
concentrations (Fig. 4). Instead, incremental changes in the
ATP/ADP ratio are predicted to provide for incremental
changes in hydrolytic activity, supporting the idea that ATP
turnover is highly tunable in this system.
Summary and Conclusions—In summary, our data support a
mechanism in which Rca ring asymmetry plays a critical func-
tional role. Our kinetic data, collected under conditions consis-
tent with an average hexameric state, suggest a minimum of
three different classes of sites. ATP hydrolysis appears to be
activated substantially by the cooperative binding of a second
Mg2ϩ ion. The mismatch in assembly stoichiometries of Rca
and Rubisco may have functional relevance. For example, Rca
hexameric assemblies exhibiting 2-fold symmetry could func-
tion by engaging only two or four subunits with Rubisco. We
hope that the development of a mechanistic framework will
serve as a starting point from which in vivo regulation of
Rubisco activation can be understood.
Knowledge of the Hill coefficient under physiologically rele-
vant conditions appears critical when analyzing “energy
charge” curves in relation to product inhibition (Fig. 4, a–c).
Although in this work we have measured Hill coefficients of 1.9
and 2.5, we cannot exclude the possibility that under different
conditions the Rca assemblies will operate with a higher degree
of positive cooperativity. For example, nH was shown to vary
between 2 and 4 for ClpX (50), supporting functional models
that involve conformational switching in the four-nucleotide
state (64). It is feasible that for tobacco Rca, the measured Hill
coefficients appear depressed due to co-existing populations of
oligomeric species with less than six subunits or due to coexist-
ing populations of larger supramolecular assemblies that are
less ATPase active. Regardless, the observed positive subunit
cooperativity supports a process that involves pairwise switch-
ing in an either coordinated or stochastic fashion (nH Ϸ2) but
excludes a fully concerted process (nH Ϸ6). We suggest that
high ATP and high magnesium set ring-like assemblies into a
Rubisco reactivation-competent state. Once the catalytic state
has been reached, the occupation of a fraction of active sites
with ADP may result in a cooperative system capable of high
turnover rates in a steady-state fashion (Fig. 5). Based on our
kinetic data, we estimate that each Rca subunit hydrolyzes
about 10 ATP molecules within 30 s, followed by a sharp
decrease in velocity. If a 30-s steady-state turnover of one hex-
amer were coupled to one Rubisco activation event, the entire
process would require the hydrolysis of 60 ATP molecules.
Author Contributions—S. H., J. N. H., K. L., and M. T. H. acquired,
analyzed, and interpreted the experimental data. R. M. W. developed
the research strategy, directed the project, and wrote the manuscript.
All authors reviewed the results and approved the final version of the
manuscript.
Acknowledgments—Tobacco Rubisco preparations, ribulose bisphos-
phate (RuBP), and diphosphoglycerate mutase (dPGM-streptavidin)
were the generous gifts from Dr. Michael Salvucci, United States
Department of Agriculture-ARS.
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24236 JOURNAL OF BIOLOGICAL CHEMISTRY
VOLUME 290•NUMBER 40•OCTOBER 2, 2015

Bioenergetics:
Regulation of Ribulose-1,5-bisphosphate
Carboxylase/Oxygenase (Rubisco)
Activase: PRODUCT INHIBITION,
COOPERATIVITY, AND MAGNESIUM
ACTIVATION
Suratna Hazra, J. Nathan Henderson, Kevin
Liles, Matthew T. Hilton and Rebekka M.
Wachter
J. Biol. Chem. 2015, 290:24222-24236.
doi: 10.1074/jbc.M115.651745 originally published online August 17, 2015
Access the most updated version of this article at doi: 10.1074/jbc.M115.651745
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