active-site carbamate formation and reaction-intermediate-analog

5
Proc. NatI. Acad. Sci. USA Vol. 81, pp. 3660-3664, June 1984 Biochemistry Active-site carbamate formation and reaction-intermediate-analog binding by ribulosebisphosphate carboxylase/oxygenase in the absence of its small subunits (cyanobacteria/Synechococcus/protein subunit interactions) T. JOHN ANDREWS* AND BETH BALLMENT Australian Institute of Marine Science, PMB No. 3, IMC, Townsville, QLD 4810, Australia Communicated by Olle Bjorkman, February 29, 1984 ABSTRACT Even though depleted of more than 90% of its small subunits, ribulose 1,5-bisphosphate carboxylase/oxy- genase from Synechococcus ACMM 323 still formed a stable complex with 2-carboxyarabinitol 1,5-bisphosphate from which exchange of the activator CO2 molecule was prevented. The stoichiometry between nonexchangeable CO2 and large subunits was unchanged regardless of the presence or absence of small subunits. The small-subunit-depleted enzyme was also "activated" by exposure to CO2 and Mg2+, although it was necessary for the small subunits to be bound before this "acti- vation" could be expressed. Binding of small subunits oc- curred rapidly, its rate depending on subunit concentration. The initial rate of "activation" was not slowed in the absence of small subunits but its extent at equilibrium was reduced. These observations are not consistent with an obligate role for the small subunits in the activation process. Their necessity in catalysis must stem from a more subtle involvement in the cat- alytic mechanism itself. The enzyme ribulose 1,5-bisphosphate carboxylase/oxygen- ase (RuBisCO) (EC 4.1.1.39), which catalyzes the initial re- action of the Calvin C02-fixing cycle of photosynthesis, also catalyzes the first reaction of this cycle's apparently waste- ful photorespiratory appendage (1). RuBisCO from most sources is a hexadecamer consisting of eight 52-kilodalton (kDa) large subunits (L) and eight 12- to 18-kDa small sub- units (S). Probes that label the catalytic site have been shown, without exception, to become attached to residues of L whose primary sequence shows remarkable homology be- tween sources as phylogenetically divergent as cyanobac- teria, green algae, and higher plants (2). At least part of the active site must, therefore, reside on L. The function of S is uncertain. The existence of at least one RuBisCO that lacks it-i.e., the Rhodospirillum rubrum enzyme, which has an L2 subunit structure (3)-might lead to the idea that S does not have a catalytic function. However, the sequence of L from this RuBisCO is very different from that of the L8S8 enzymes (4) and the functionality of S may have become in- corporated into L during the evolution of R. rubrum. Ac- cording to this logic, the L2 structure might be considered more advanced than the L8S8, rather than the reverse. Re- versible removal of S from the L8S8 enzymes from some cyanobacteria has been reported (5, 6). In the case of the RuBisCO from Synechococcus ACMM 323 it was shown that S was absolutely essential for catalytic function (7). The well-studied activation of the enzyme (E) RuBisCO by CO2 and a divalent metal ion (M2+), such as Mg2+ (Scheme 1), was reviewed recently by Miziorko and Lorimer (2). slow m2 E-NH+ + E-NH2 + aco E-NH-C-00 + M2 Inactive ,- fast - - . ~~~~1F~ - Active E-NH-COO *M2+ Scheme 1. The activating CO2 molecule, designated aCO2 to differenti- ate it from the CO2 molecule that acts as substrate for the carboxylation reaction, TC02 (8), reacts with the E-amino group of lysine-201 of L [the spinach numbering sequence (9) is used] to form a carbamate, which is stabilized by the sub- sequent binding of the metal ion. This is a freely reversible process and the proportion of active enzyme (i.e., E- aCO,__M2+) at equilibrium depends on the pH and CO2 and M2+ concentrations. The position of the equilibrium of Scheme 1 is also influenced by the binding of a variety of phosphorylated compounds to the ribulose 1,5-bisphosphate (Rbu-P2) binding site, and some of these interactions may be important in regulating the level of activation in vivo. These compounds exert their effect by hindering the exchange of aCO2 and M2+, thus indicating that the activation reactions occur at the catalytic site (10-12). Most notable among these compounds is the reaction-intermediate analog 2-carboxyar- abinitol 1,5-bisphosphate (CA-P2), which gives rise to a qua- ternary E-aCO2T M2+_CA-P2 complex that is remarkably stable, even in the absence of the unbound ligands (13-16). These activation reactions provided an opportunity to study the effects of. S on a function of RuBisCO other than catalysis. Results showed that S is not obligatorily required for activation and CA-P2 binding, suggesting a more subtle role for S in the catalytic reaction itself. MATERIALS AND METHODS Materials. RuBisCO was purified from Synechococcus strain ACMM 323 (formerly RRIMP N1) by published proce- dures (7). The specific activity was 4.4 pAmol min1 per mg of protein at 25TC. Tetrasodium Rbu-P2 was purchased from Sigma or Calbiochem. Tetralithium CA-P2 (lactone form) was kindly provided by G. H. Lorimer and was dissolved in 50 mM Tris HCI, pH 9, and stored overnight at room tem- perature before use. Methods. L and S contents of RuBisCO preparations were measured by the high-performance-gel-filtration procedure Abbreviations: Rbu-P2, D-ribulose 1,5-bisphosphate; RuBisCO, Rbu-P2 carboxylase/oxygenase; L and S, large and small subunits of RuBisCO; CA-P2, 2-carboxyarabinitol 1,5-bisphosphate; kDa, ki- lodalton(s). *Present address: E. I. du Pont de Nemours & Co., Central Re- search & Development Department, Experimental Station 402- 2125, Wilmington, DE 19898. 3660 The publication costs of this article were defrayed in part by page charge payment. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. §1734 solely to indicate this fact.

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Page 1: Active-site carbamate formation and reaction-intermediate-analog

Proc. NatI. Acad. Sci. USAVol. 81, pp. 3660-3664, June 1984Biochemistry

Active-site carbamate formation and reaction-intermediate-analogbinding by ribulosebisphosphate carboxylase/oxygenase in theabsence of its small subunits

(cyanobacteria/Synechococcus/protein subunit interactions)

T. JOHN ANDREWS* AND BETH BALLMENTAustralian Institute of Marine Science, PMB No. 3, IMC, Townsville, QLD 4810, Australia

Communicated by Olle Bjorkman, February 29, 1984

ABSTRACT Even though depleted of more than 90% ofits small subunits, ribulose 1,5-bisphosphate carboxylase/oxy-genase from Synechococcus ACMM 323 still formed a stablecomplex with 2-carboxyarabinitol 1,5-bisphosphate fromwhich exchange of the activator CO2 molecule was prevented.The stoichiometry between nonexchangeable CO2 and largesubunits was unchanged regardless of the presence or absenceof small subunits. The small-subunit-depleted enzyme was also"activated" by exposure to CO2 and Mg2+, although it wasnecessary for the small subunits to be bound before this "acti-vation" could be expressed. Binding of small subunits oc-curred rapidly, its rate depending on subunit concentration.The initial rate of "activation" was not slowed in the absenceof small subunits but its extent at equilibrium was reduced.These observations are not consistent with an obligate role forthe small subunits in the activation process. Their necessity incatalysis must stem from a more subtle involvement in the cat-alytic mechanism itself.

The enzyme ribulose 1,5-bisphosphate carboxylase/oxygen-ase (RuBisCO) (EC 4.1.1.39), which catalyzes the initial re-action of the Calvin C02-fixing cycle of photosynthesis, alsocatalyzes the first reaction of this cycle's apparently waste-ful photorespiratory appendage (1). RuBisCO from mostsources is a hexadecamer consisting of eight 52-kilodalton(kDa) large subunits (L) and eight 12- to 18-kDa small sub-units (S). Probes that label the catalytic site have beenshown, without exception, to become attached to residues ofL whose primary sequence shows remarkable homology be-tween sources as phylogenetically divergent as cyanobac-teria, green algae, and higher plants (2). At least part of theactive site must, therefore, reside on L. The function of S isuncertain. The existence of at least one RuBisCO that lacksit-i.e., the Rhodospirillum rubrum enzyme, which has anL2 subunit structure (3)-might lead to the idea that S doesnot have a catalytic function. However, the sequence of Lfrom this RuBisCO is very different from that of the L8S8enzymes (4) and the functionality of S may have become in-corporated into L during the evolution of R. rubrum. Ac-cording to this logic, the L2 structure might be consideredmore advanced than the L8S8, rather than the reverse. Re-versible removal of S from the L8S8 enzymes from somecyanobacteria has been reported (5, 6). In the case of theRuBisCO from Synechococcus ACMM 323 it was shownthat S was absolutely essential for catalytic function (7).The well-studied activation of the enzyme (E) RuBisCO

by CO2 and a divalent metal ion (M2+), such as Mg2+(Scheme 1), was reviewed recently by Miziorko and Lorimer(2).

slow m2E-NH+ + E-NH2 + aco E-NH-C-00 + M2

Inactive ,- fast-- . ~~~~1F~-Active

E-NH-COO *M2+

Scheme 1.

The activating CO2 molecule, designated aCO2 to differenti-ate it from the CO2 molecule that acts as substrate for thecarboxylation reaction, TC02 (8), reacts with the E-aminogroup of lysine-201 ofL [the spinach numbering sequence (9)is used] to form a carbamate, which is stabilized by the sub-sequent binding of the metal ion. This is a freely reversibleprocess and the proportion of active enzyme (i.e., E-aCO,__M2+) at equilibrium depends on the pH and CO2 andM2+ concentrations. The position of the equilibrium ofScheme 1 is also influenced by the binding of a variety ofphosphorylated compounds to the ribulose 1,5-bisphosphate(Rbu-P2) binding site, and some of these interactions may beimportant in regulating the level of activation in vivo. Thesecompounds exert their effect by hindering the exchange ofaCO2 and M2+, thus indicating that the activation reactionsoccur at the catalytic site (10-12). Most notable among thesecompounds is the reaction-intermediate analog 2-carboxyar-abinitol 1,5-bisphosphate (CA-P2), which gives rise to a qua-ternary E-aCO2T M2+_CA-P2 complex that is remarkablystable, even in the absence of the unbound ligands (13-16).These activation reactions provided an opportunity to

study the effects of.S on a function of RuBisCO other thancatalysis. Results showed that S is not obligatorily requiredfor activation and CA-P2 binding, suggesting a more subtlerole for S in the catalytic reaction itself.

MATERIALS AND METHODSMaterials. RuBisCO was purified from Synechococcus

strain ACMM 323 (formerly RRIMP N1) by published proce-dures (7). The specific activity was 4.4 pAmol min1 per mgof protein at 25TC. Tetrasodium Rbu-P2 was purchased fromSigma or Calbiochem. Tetralithium CA-P2 (lactone form)was kindly provided by G. H. Lorimer and was dissolved in50 mM Tris HCI, pH 9, and stored overnight at room tem-perature before use.Methods. L and S contents of RuBisCO preparations were

measured by the high-performance-gel-filtration procedure

Abbreviations: Rbu-P2, D-ribulose 1,5-bisphosphate; RuBisCO,Rbu-P2 carboxylase/oxygenase; L and S, large and small subunitsof RuBisCO; CA-P2, 2-carboxyarabinitol 1,5-bisphosphate; kDa, ki-lodalton(s).*Present address: E. I. du Pont de Nemours & Co., Central Re-search & Development Department, Experimental Station 402-2125, Wilmington, DE 19898.

3660

The publication costs of this article were defrayed in part by page chargepayment. This article must therefore be hereby marked "advertisement"in accordance with 18 U.S.C. §1734 solely to indicate this fact.

Page 2: Active-site carbamate formation and reaction-intermediate-analog

Proc. NatL. Acad Sci. USA 81 (1984) 3661

described previously (7) except that 206-nm detection wasused. In calculating the molar concentrations of L, a pro-tomer mass of 51.5 kDa was assumed. This value reflects thesequence of maize L, assuming that the NH2 terminus of themature subunit is alanine-15 (17). A similar protomer masswas derived with the same assumption from the sequence ofL from Synechococcus PCC 6301 (18). This value is signifi-cantly smaller than that measured for L from most sourcesby physical methods. A protomer mass of 14 kDa for S wascalculated from its retention time during high-performancegel filtration by calibrating against standard proteins. NativeSynechococcus RuBisCO concentrations and Rbu-P2 car-boxylase activities were measured as described previously(5, 7). S was stripped from the L8 core of SynechococcusRuBisCO by precipitation at pH 5.35 in 10 mM phosphate/acetate buffer containing 500 mM NaCi and 1 mM EDTA asdescribed previously (7). One or two precipitations wereused as indicated.

Analysis of Kinetics of Activation. Activation of RuBisCO(Scheme 1) follows an exponential time course (19) and maybe represented by the equation

a, = ae(l - e-kl),

ca0= 9o

I-

CD00

0-1x

c'J0 30~00 9

E0

°26.e

cn 3

0

-Jo

[1]

in which a, and ae are the extents of activation at time t andat equilibrium, respectively, and k is the pseudo-first-orderrate constant for activation. Since activation of the Synecho-coccus enzyme is very rapid, further activation occurringduring the catalytic phase of the reaction following additionof Rbu-P2 cannot be ignored. During this phase, both ae andk may be different from the corresponding parameters pre-vailing during the activation phase and are designated a' andk'. The molar quantity of CO2 fixed during a catalytic phaselasting 30 s, m,, is thus given by

30

mt, = [a, + (a' - a,)(1 - e-k ')]dt', [2]e

in which t' is the time during the catalytic phase. By integrat-ing and substituting for a,, it may be shown that

m, = A - Be-ki [3]

2 4 6 8 10 12

Fraction

).9

).6

coI-

I0

E.9

cV0

0

6 'rt

.3

FIG. 1. Binding of nonexchangeable "CO2 by native (a) and S-depleted (b) RuBisCO in the presence of Mg2' and CA-P2. Nativeenzyme was equilibrated with a C02-free buffer solution containing25 mM Hepes-NaOH and 1 mM EDTA, pH 7.7, and adjusted to a

final concentration of 12 ,.M L protomers by ultrafiltration using an

Amicon YM30 membrane. S-depleted enzyme was prepared by asingle mild-acid precipitation and dissolved in the same buffer to a

final concentration of 9 ,uM L protomers. To 500 i.d of each enzymesolution was added 50 ,l of 0.5 M NaH14CO3 (3800 dpm nmol-1)and 10 ,l of 1 M MgCI2. After 10 min at room temperature, 10 ,ul of 5mM CA-P2 was added, giving a 7- to 10-fold excess of CA-P2 overactive sites. After a further 15 min, the solutions were applied to 7 x200 mm columns of Sephadex G-25 medium equilibrated and elutedwith 25 mM Hepes-NaOH/1 mM EDTA/100 mM NaHCO3, pH7.7. Four-drop fractions were collected and assayed for 14C trappedby alkali (x), L (o), and S (e). The mean of the 14CO2/L molar ratios(o) for the three peak fractions is represented by the broken line.

in which

A = a[30 - (1 - e-30k)/kI]+ ae(1- e3k)/k' [4]

and

B = ae(l - &30k)/kI. [5]

RESULTS

Formation of a Stable E-aCO,.Mg2+_CA-P2 Complexin the Absence of S. In the presence of Mg2+ and CA-P2,14CO2 was bound to the activator site of SynechococcusRuBisCO. The stoichiometry of this binding was 0.74 ± 0.02(range) mol of CO2 per mol of L protomer and was the sameregardless of whether native enzyme or enzyme partially(Fig. 1) or nearly completely (Table 1) depleted of S wasused. The bound 14CO2 did not exchange with 100 mMNaHCO3 present in the column elution buffer, thus ensuringthat binding occurred at the activator site, where it was pre-vented from exchanging by the presence of CA-P2. Further-more, no binding occurred in the absence of Mg2+ and CA-P2 (Table 1). When Mg2' alone was omitted, no binding tothe S-depleted preparation was observed, but a variable de-

gree of binding to the native enzyme still occurred (Table 1).Apparently, in the case of the native enzyme but not the S-depleted enzyme, added metal ions were not required toform a complex between enzyme, C02, and CA-P2 that wasstable enough to partially survive passage through the gelfiltration column (about 2 min in duration).

Activation in the Absence of S. For convenience, the term"activation" is retained to describe the process representedby Scheme 1 even though it does not result in catalyticallyactive enzyme if S is missing. We examined the possibilitythat activation occurred in the absence of S by observing thetime courses of activation of S-depleted, reconstituted, andnative enzyme preparations at low CO2 concentration, atwhich the rate of activation was slow enough to be mea-

sured. When S was absent during activation, it was addedsimultaneously with the substrate, Rbu-P2, to initiate thecatalytic phase of the reaction. The results (Fig. 2, Table 2)showed that Synechococcus RuBisCO activated very rapidlyeven at low CO2 concentration, confirming earlier data (5),and that activation did indeed occur in the absence of S.However, when S was missing during the activation phase,the extent of activation at equilibrium, though not its initialrate, appeared considerably reduced. Activation was so rap-id that, in the absence of carbonic anhydrase, its initial ratewas limited by the interconversion of HCO-, used to initiateactivation, to C02, the species effective in activation (19).

r I

a0

0

-b0

0.

0~~~~0

V/i 0

L /-W I I 1 A

r

-

I

L c

Biochemistry: Andrews and Ballment

Page 3: Active-site carbamate formation and reaction-intermediate-analog

3662 Biochemistry: Andrews and Ballment

Table 1. Effect of Mg2' and CA-P2 on binding ofnonexchangeable '4CO2 by native and S-depletedRuBisCO

Nonexchangeable 1'4CO2,mol 14C per mol of L

protomers

Omission Native S-depletedNone 0.74 0.73Mg2+ 0.45, 0.28 0Mg2+ and CA-P2 0

S-depleted enzyme was prepared by two successive mild-acid pre-cipitations. Its S content was less than 8% of that of the native en-zyme. The binding experiments were carried out as described forFig. 1 except that Mg2+ and CA-P2 were selectively omitted duringthe activation phase as indicated. The effluent of the Sephadex col-umn was continuously monitored at 280 nm, enabling collection ofthe protein peak eluting immediately after the void volume as a sin-gle fraction. This fraction was assayed for L, S, and 14C trapped byalkali.

This led to nonexponential time courses with initial lagswhen carbonic anhydrase was omitted (data not shown).

Kinetics of Reconstitution. The rate at which S-depletedenzyme recombined with isolated S to yield catalytically ac-tive enzyme was studied by fully activating an S-depletedpreparation at high CO2 concentration and then observingthe time course of catalysis initiated by the simultaneous ad-dition of S and Rbu-P2 (Fig. 3). High CO2 concentrationswere used to promote activation even if its equilibrium wasunfavorable in the absence of S. Furthermore, since activa-tion of Synechococcus RuBisCO is almost instantaneouswhen CO2 is saturating (5), any further potential for activa-tion occurring on reconstitution would be realized very rap-idly. Therefore, the contribution of activation to any lag ob-served would be insignificant and, presumably, such a lagmust reflect the reconstitution process itself. A lag was in-deed observed (Fig. 3). Its effective duration, calculatedfrom the intercept on the x-axis of the extrapolated linearportion of the progress curve, was strongly dependent on theconcentrations of the subunits, being 54 s at 27 nM L, 12.5 sat 110 nM L, and 2.4 s at 440 nM L (the S/L molar ratiobeing held constant at 1.2 in all cases). Since the subunitconcentrations used in the activation experiments (Fig. 2)were approximately at the highest concentrations used here,the effect of this reconstitution lag on the observed activa-tion kinetics would not have been significant. When recon-stitution occurred before activation, the subsequent catalyticreaction showed no lag (Fig. 3).Two additional features are discernible in the reconstitu-

tion data (Fig. 3). First, the specific activity of the reconsti-tuted enzyme was about 30% lower when measured at thelowest subunit concentrations (Fig. 3c), compared to thehigher concentrations (Fig. 3 a and b). This presumably re-flects the equilibrium of the reconstitution process and isconsistent with a Kd in the nanomolar range (7). Second, therates achieved subsequent to the reconstitution lag when Swas added at zero time were smaller than the catalytic ratesof the enzyme reconstituted before assay, and the discrepan-cy became progressively larger as the subunit concentrationswere lowered. However, this might be only a transient phe-nomenon because the latter assays showed a slight, but con-sistent, tendency to decelerate and it is possible that, aftersufficient time, the catalytic rates might have become identi-cal regardless of the order of addition of S. The latter obser-vations would be understandable if Rbu-P2 loosened thebinding of S-i.e., increased its Kd-thereby decreasing theproportion of reconstituted enzyme at equilibrium, particu-larly at low subunit concentrations. Furthermore, whenRbu-P2 was added after reconstitution had reached equilibri-

12

0 .90

3j0 I. .

40 80 120 160

Time, sFIG. 2. Activation of RuBisCO in the presence (o) and absence

(e) of S. S-depleted enzyme was prepared by two successive precip-itations. To avoid contamination of the isolated S preparation withtraces of unprecipitated L, which would complicate this experiment,complete precipitation was ensured by precipitating at the lower-than-usual pH of 5.1. The resultant S-containing supernatant fromthe first precipitation was devoid ofL and catalytic activity. Howev-er, a little more irreversible denaturation of the pelleted L occurredat this pH (7). The resultant preparation contained less than 8% ofthe native S content and its specific activity in terms ofL protomers,measured by the standard assay, was 3% of that of the native en-zyme in the absence of added S and 51% after full saturation with S.The time course of activation was measured at 20°C in 4-ml septum-capped vials containing 0.5 ml of a C02-free buffer solution contain-ing 40 mM N,N'-bis(2-hydroxyethyl)glycine-NaOH at pH 7.7, 16mM MgCl2, bovine serum albumin at 0.1 mg-ml-', and bovine car-bonic anhydrase at 0.1 mg ml-1. Both solution and head space wereflushed with N2. For the experiment described by o, the S-depletedenzyme (13 ,ug) and isolated S (4.4 &g) were added to this solution,giving a 1.3-fold molar excess of S over L and, after at least 5 min,activation was initiated by adding NaH14CO3 (3900 dpm'nmol-') to0.5 mM. At the indicated times, Rbu-P2 was added to 0.4 mM, fol-lowed by 0.15 ml of formic acid 30 s later. For the experiment de-scribed by e, isolated S was omitted during the activation phase andadded simultaneously with the Rbu-P2. The presence or absence ofS had negligible effect on the pH of the solution during the activationphase. Acidified samples were dried at 80°C and 14C was determinedby scintillation counting. When isolated S was omitted altogether,the activity recorded after activation to equilibrium was 0.97 nmolper 30 s per mg of L. The solid lines show the best fit of the data toEq. 3.

um in its absence, slow loss of activity would be expecteduntil the new equilibrium position was reached.

DISCUSSIONThe observations that a stable E-aCO2 Mg2+-CA-P2complex was formed by Synechococcus RuBisCO in the par-tial (Fig. 1) or nearly complete (Table 1) absence of S andthat the stoichiometry between bound CO2 and L remainedunchanged regardless of S content show unequivocally thatthe chemistry of activation and reaction-intermediate-analogbinding does not depend on S in any obligate manner. There-fore, the mechanisms underlying the requirements for S inthe catalytic reaction (7) must be sought elsewhere.There could be several trivial reasons for the observed

substoichiometric binding of aCO2 (i.e., 0.74 mol of CO2 permol of L protomer rather than the expected ratio of 1.0).Some fraction of the purified enzyme preparation may havebeen denatured during purification to the extent that it was

Proc. NatL Acad Sci. USA 81 (1984)

Page 4: Active-site carbamate formation and reaction-intermediate-analog

Proc. Nat. Acad. Sci. USA 81 (1984) 3663

Table 2. Kinetic parameters for the activation of RuBisCO in the presence and absence of S

A, B, ae, ae,Enzyme nmol(30 s)' nmol-(30 s)-' pmols1 pmol s' k, k',form* per mg of L per mg of L per mg of L per mg of L s- x 102 s- 1x 102

Native 35.4 + 0.6 29.0 ± 0.6 1180 1180 2.34 ± 0.14 1.37Reconstituted 12.5 ± 0.2 10.1 ± 0.3 416 416 4.28 ± 0.31 1.44S-depleted 4.9 ± 0.1 2.5 ± 0.2 104 (75)t 416 24.36 ± 4.99 1.50

The parameters A, B. and k were estimated (± SD) by fitting the data sets presented in Fig. 2 and a further set for nativeenzyme to Eq. 3 by means of nonlinear regression (PAR program of the BMDP package produced by the Health SciencesComputing Facility, University of California, Los Angeles). The parameters ae, a', and k' were then estimated as follows.In the cases of the native and reconstituted preparations, the only difference in conditions between the activation andcatalytic phases of the reaction was the presence of Rbu-P2 during the latter phase. It was shown previously that Rbu-P2 isa neutral effector of the activation of Synechococcus RuBisCO-i.e., its presence during activation at subsaturating CO2concentrations did not alter the extent of activation at equilibrium (5). Therefore, from Eq. 4, for the native enzyme andpresumably for the reconstituted enzyme also, a' = ae = A/30 and therefore k' may be calculated by using Eq. 5. Condi-tions during the catalytic phases of the experiments in which the reconstituted and S-depleted preparations were activatedwere similar since the final reaction mixtures contained precisely the same constituents. Therefore a' must be the same forthe two experiments and k' and ae for the experiment in which S-depleted enzyme was activated can thus be calculated byusing Eqs. 4 and 5.*The enzyme form listed is that which prevailed during activation.tTraces of S still remaining after subunit separation caused the specific activity in the absence of added S to be about 3% ofthat of the native enzyme (Fig. 2 legend). While this trace of activity is negligible in most circumstances and has littleeffect on the estimate of k for activation in the absence of S, its effect on ae is significant because the latter parameter wasso much reduced when S was absent. Since these traces of S were present during the activation phase, it may be presumedthat activation of this small portion of the enzyme would have occurred with kinetics similar to that of the native orreconstituted preparations. Therefore the ae parameter for activation in the absence of S may be corrected for the activitymeasured in this control. The corrected value is shown in parenthesis.

no longer capable of activation and quaternary complex for-mation. Hall et al. (16) observed this phenomenon with spin-ach RuBisCO. Alternatively, one of the assumptions onwhich calibration of the high-performance-gel-filtration tech-nique for estimation of L and S is based (7) may be in error.The most obvious uncertainty is the absorption coefficientof the native enzyme. A value of 1.26 ml-mg-1 cm-l was as-sumed, based on protein mass measured by the unorthodoxtechnique of amino acid analysis because of its scarcity (7).Ifthe widely used absorption coefficient of1.64 ml-mg- '-cm -1for spinach RuBisCO (20) is used, the stoichiometry be-comes 0.97.While these complex-formation experiments show that S

is not required for activation, they are essentially qualitativein nature. CA-P2 binds to the active site of RuBisCO extraor-dinarily tightly (Kd = 0.3 pM*) and decreases the rates ofdissociation of aCO2 and the metal ion involved in activationby six to seven orders of magnitude (G. H. Lorimer andJ. V. Schloss, cited in ref. 2). Looser binding of CA-P2 to theS-depleted enzyme would therefore pass unnoticed even ifthe Kd was several orders of magnitude greater than that ofthe native enzyme. In the absence of added metal ions, how-ever, binding of aCO2 and CA-P2 to spinach RuBisCO ismore freely reversible, although the complex is still stableenough to be recovered partially after gel filtration (14, 15).While a similar complex was readily demonstrable with na-tive Synechococcus RuBisCO in the absence of added metalions, it could not be detected with S-depleted enzyme (Table1). This observation is consistent with looser binding of CA-P2 in the absence of S.

Studies of the kinetics of formation of activated enzymeconfirmed that the activation reactions (Scheme 1) do indeedproceed to a measurable extent in the absence of both S andCA-P2 and showed that the S-depleted preparation activatedjust as rapidly as the reconstituted enzyme (Fig. 2, Table 2).These experiments were complicated because any activa-tion, as defined by Scheme 1, that occurred in the absence ofS could not be expressed until the activated complex hadbound S. Fortunately, binding of S was nearly instanta-neous, provided the L and S concentrations were high

*Lorimer, G. H., 12th International Congress of Biochemistry,Aug. 15-21, 1982, Perth, Western Australia, abstr. SYM 043-3.

enough (Fig. 3), and therefore S could be added to the de-pleted preparation, after activation in its absence, at the be-ginning of the catalytic phase.

4 10. CIa b

-~~~~~2 7

60 00 30 6

a~~~~~~~.X ~~~~7.50

5.0 - ~ ~~

2.5-

0--0 100 200 300

Time, S

FIG. 3. Time courses of the catalytic reaction of RuBisCO underC02-saturated conditions. S-depleted enzyme and isolated S prepa-rations were prepared as for Fig. 2. Reactions were carried out at20°C in magnetically stirred, air-saturated buffer solution containing80'mM Hepes-NaOH at pH 7.7, 20 mM MgCl2, 40 mM NaCl, andbovine serum albumin at 0.1 mg ml-'. For the time courses de-scribed by o, the S-depleted enzyme and the isolated S preparationwere added, giving a 1.2-fold molar excess of S over L, and activat-ed for at least 10 min after addition of NaH'4CO3 (460 dpm nmol-')to 70 mM. Catalysis was then initiated by adding Rbu-P2 to 0.4 mM.At the stated times thereafter, 0.3-ml aliquots were removed andadded to 1 ml of 50%' formic acid. For e, isolated S was omittedduring the activation phase and added simultaneously with the Rbu-P2. Acidified samples were dried and their radioactivities were mea-sured as for Fig. 2. The final concentrations of L protomers in thethree experiments were 440 nM (a), 110 nM (b), and 27 nM (c). Notethe change of scales for c.

Biochemistry: Andrews and Ballment

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3664 Biochemistry: Andrews and Ballment

The difference in the extent of activation at equilibrium,ae, between the native and reconstituted preparations (Table2) is trivial, mostly reflecting irreversible denaturation in-curred during separation of the subunits. (The small, but sig-nificant, increase in k after reconstitution may be anotherindication of damage to the reconstituted enzyme.) Howev-er, when S was absent during activation, ae was reduced to1/6th of the value for the reconstituted enzyme. Therefore,the overall equilibrium of the activation reactions (Scheme 1)must lie much further to the left when S is missing. The 6-fold greater value of k in the absence of S is consistent withthe reduced value of ae. The effect of S on activation is thusreminiscent of that of Mg2+, which also increases ae for acti-vation but not its initial rate, resulting in an inverse depen-dency of k on Mg2+ concentration (19).The values of the pseudo-first-order rate constant for fur-

ther activation during the catalytic phase (k') for the threeexperiments were not significantly different (Table 2). Thisis to be expected because the compositions of the reactionmixtures were identical during this phase and it confirms theconclusion that reconstitution of S with L8 to form catalyt-ically active enzyme must have occurred virtually instanta-neously under the conditions used. Asami et al. (6) also ob-served instantaneous reconstitution of the Aphanothece ha-lophytica enzyme. The subunit concentrations in theirexperiments were considerably higher than those used here.

It has been suggested that one of the functions of S is toaccelerate activation (21). This theory was based on compar-isons of the activation rates of RuBisCOs that naturally lackS (from Rhodospirillum rubrum and form II from Rhodo-pseudomonas sphaeroides) with those that have it (fromspinach and form I from Rhodopseudomonas sphaeroides).However, as pointed out by Miziorko and Lorimer (2), thesedifferences in activation rate could just as well be attributedto differences in L. Our present results rule out such a rolefor S in Synechococcus RuBisCO since it activates at least asrapidly in the absence of S as in its presence (Fig. 2). Rather,the role of S in activation is in stabilizing the E-CO2-Mg2+complex, perhaps by decelerating deactivation. However, itis clear that this is not an obligatory role and would not, byitself, make S essential for catalysis. Even at high CO2 andMg2+ concentrations where activation in the absence of Swould be promoted to higher levels than the level reported inFig. 2, catalytic activity was directly proportional to S con-tent, indicating that S is absolutely required for catalysis (7).Although our data are not consistent with an obligate role

for S in the chemistry of activating carbamate formation,they do not preclude the possibility that S mediates someaspect of the complex and incompletely understood processwhereby the activation level of RuBisCO is regulated in vivo.For instance, it is likely that S influences the interactionswith the Rbu-P2-binding site of phosphorylated effectorsthat regulate the level of activation-i.e., the position of theequilibrium of Scheme 1 (10-12). Interactions of S with thesubstrate-binding site are indicated by the effect of S itselfon this equilibrium, the looser binding of CA-P2 in the ab-sence of S and Mg2+, and the inference that Rbu-P2 mayhave an effect on the binding of S.

The obligate function of S must be sought in the catalyticmechanism itself. Broadly stated, the most likely possibilityis that binding of S is required to induce a catalytically com-petent conformation in L. However, the influence of S mustbe rather subtle. Clearly, when S is missing, the active sitestill bears enough resemblance to its native condition to forma stable E-aCO2_Mg2+ CA-P2 complex. Miziorko (13)suggested that binding of the carboxyl group of CA-P2 to thesCO2-binding site was implicated in the very tight bindingachieved by this analog. If so, its tight binding to S-depletedRuBisCO may provide evidence that the sCO2-binding site isalso on L. However, more quantitative studies of the tight-ness of binding of CA-P2 in the absence of S will be requiredbefore this can be established. For the present, we do notconsider that the interesting possibility that the binding sitefor the gaseous substrate resides on S has been excluded.

We thank F. Gillan for assistance with the mathematical treatmentof the kinetics of activation. This is contribution no. 231 from theAustralian Institute of Marine Science.

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Proc. NatL Acad Sci. USA 81 (1984)