oxygenase

Biochimica etBiophysicaActa, 743 (1983) 207-211

207

Elsevier Biomedical Press BBA31525

A S H A R P T R A N S I T I O N IN ACTIVITY AND C O N F O R M A T I O N OF TOBACCO RIBULOSE-I,5-BISPHOSPHATE CARBOXYLASE/OXYGENASE MASASHI MIYANO

Central Research Institute, Japan Tobacco and Salt Public Corporation, 6- 2 Umegaoka, Midori- ku, Yokohama 227 (Japan) (Received July 6th, 1982) (Revised manuscript received November 30th, 1982)

Key words: Ribulosebisphosphate carboxylase; Conformational change," Viscosity," Temperature sensitivity," (N. tabacum)

The temperature responses of the carboxylase activity and the reduced viscosity of crystalline ribulose-l,5bisphosphate carboxylase/oxygenase (EC 4.1.1.39) from tobacco leaves were measured. The Arrhenius plots of initial rates of carboxylase activity of the enzyme were biphasic, with a break at 16°C when the enzyme solution, stored at 4°C with magnesium and bicarbonate ions, was warmed to the assay temperature. The temperature response of the reduced viscosity of this enzyme solution was also biphasic, with a sharp transition at 15°C. However, these states of the enzyme were not distinguished by analytical sedimentation. These results show that a conformational transition, which affects not only a hydrodynamic property but also the carboxylase activity of ribulose-l,5-bisphosphate carboxylase/oxygenase, occurs around 15°C. The temperature transition in carboxylase activity disappeared with a 1-h preincubation of the protein at 30°C with magnesium and bicarbonate ions.

Introduction Ribulose-l,5-bisphosphate carboxylase/ oxygenase (EC 4.1.1.39) is responsible for photorespiration as well as photosynthetic fixation of carbon dioxide in green plants [1]. This bifunctional enzyme catalyzes either the conversion of ribulose 1,5-bisphosphate and carbon dioxide to two molecules of 3-phosphoglycerate, or the conversion of ribulose 1,5-bisphosphate and oxygen to equimolar amounts of 3-phosphoglycerate and 2phosphoglycolate. Both reactions are irreversible, and this enzyme is thought to be one of the regulatory photosynthetic enzymes,. This hexadecameric protein is composed of eight large (54-kDa) and eight small (14-kDa) subunits, and this complex quaternary structure is presumed to be related to an undefined regulatory function. As many authors have reported [1-6,15], this enzyme is sensitive to solvent conditions such as 0167-4838/83/0000-0000/$03.00 © 1983 Elsevier Science Publishers

temperature, pH and salt concentration as well as to several metabolic intermediates. Ribulose-l,5bisphosphate carboxylase/oxygenase has been reported to be a reversible cold-labile enzyme [2,3,5]. Although many cold-labile enzymes with oligomeric structures show drastic physical changes such as dissociation and association of subunits as a result of temperature perturbation [7,8], ribulose-l,5-bisphosphate carboxylase/oxygenase does not show such a great physical change with temperature [2,3]. However, Chollet and Anderson [5] showed that a small but appreciable conformational change occurs during cold inactivation and heat reactivation of the enzyme. These results raise the question of how the conformational changes affect catalytic activity. To probe this question, more precise experiments on the physical properties of this enzyme are necessary. The present experiments deal with the physicochemical properties of tobacco enzyme

208 in relation to a temperature-induced activity change in the carboxylase reaction. Materials and Methods Ribulose 1,5-bisphosphate was synthesized as described by Horecker et al. [9] and further purified by ion-exchange column chromatography (Dowex 1 × 8, C1- form, 200-400 mesh) using 0.01 N HC1 containing 0.05 M NaC1 as the elution medium. This protocol resulted in an excellent separation of trace amounts of ADP from ribulose 1,5-bisphosphate. Sodium [14C]bicarbonate was obtained as Nal~CO3 from Amersham. All other reagents were reagent-grade and used without further purification. Crystalline ribulose-l,5-bisphosphate carboxylase/oxygenase was isolated from tobacco (Nicotiana tabacum L., cv. BY-4) leaf homogenates by a direct crystallization method [10] with minor modifications, which included: (a) the grinding buffer c o n t a i n e d 4% p o l y ( v i n y l p y r r o l i d o n e ) ; (b) fractionation with 35-50% (4°C) ( N H 4 ) 2 S O 4 w a s included after Sephadex G-25 column chromatography; and (c) dithiothreitol was used as an antioxidant and thiol-protectant instead of 2-mercaptoethanol. The enzyme used in the present experiments was twice-recrystallized. Polyacrylamide gel electrophoresis (5% gels) followed by Coomassie blue staining showed only one band when 50 #g protein were analyzed. The Schlieren pattern of the enzyme during analytical ultracentrifugation showed a single peak without any skewness (Fig. 2). These results indicate that the enzyme is sufficiently pure for the following experiments. Protein concentration was determined by ultraviolet spectroscopy using A Icm 2 7 9 n m = 14.0 (1% solution) [12], which had been calibrated by the Biuret method with bovine serum albumin as the standard. Carboxylase activity was assayed under conditions of high substrate concentration as follows: 0.5 ml enzyme solution (250-500 ~g protein) containing 100 mM Tris-HC1 (pH 8.5 at 20°C), 100 mM NaC1, 20 mM MgC12 and 10 mM NaHI4CO3 (2 Ci/mol) was preincubated for 15 min at the respective temperature. The reaction was initiated

by adding ribulose 1,5-bisphosphate (final concentration, 2 mM). After 1 min the reaction was stopped by addition of 0.2 ml 2 N HC1. Activity was expressed as acid-stable 14C-radioactivity in the medium (nmol per min per mg protein). Assay temperature was controlled by cooling an ethanol bath within _+0.03°C. The measured carboxylase activity of the enzyme obtained under this assay protocol (max. activity, around 0.1 /zmol/min per mg protein) was lower than in recent papers (0.3-0.8/zmol/min per mg protein) [3-5,13]. Analytical ultracentrifugation was performed with a Hitachi 282 Analytical Ultracentrifuge at 40 000 rpm under the following conditions: 2.7-8.7 m g / m l enzyme in 10 mM Tris-HC1 (pH 8.5 at 20°C) and 100 mM NaC1. Viscosity was measured by a cone- and platetype rotatory viscometer (Tokyo Keiki VisconicE D L ) equipped with a cooling circulator (_+ 0.01°C) at 100 rpm where the enzyme solution (0.3-15 mg/ml) contained 100 mM NaC1 and 10 mM Tris-HC1 (pH 8.5 at 20°C). The viscosity of the solution was calculated as follows: reduced viscosity, ~rea, is ~red : ( ' ~ / T ~ 0 - - 1 ) / C (dl/g), where = solution viscosity, T0 = solvent viscosity, and c = enzyme concentration (10 mg protein/ml). Intrinsic viscosity, [~]0, is extrapolated to zero concentration from ~red' In this study ~red was used mainly because of non-Newtonian viscosity of the enzyme solution. Results Paech et al. [11] reported that high concentrations of ribulose 1,5-bisphosphate may inhibit the carboxylase and oxygenase activities of the enzyme if the substrate sample contains its epimer a n d / o r degradation products. Furthermore, Kung et al. [12] and Bahr et al. [13] reported that the carboxylase and oxygenase activities of the crystalline tobacco enzyme were markedly inhibited by high Mg 2÷ concentrations (20 mM). However, under the present assay conditions 2 mM ribulose 1,5-bisphosphate did not inhibit tobacco carboxylase activity and the rate was linear over the 1-min assay period. Thus, in order to avoid limiting concentration effects with substrates, saturated substrate concentrations (20 mM MgC12, 10 mM N a H C O 3 and 2 mM ribulose 1,5-bisphosphate)

209

were used in the following activity experiments. The temperature response of the carboxylase activity of tobacco ribulose-l,5-bisphosphate carboxylase/oxygenase was measured with two different temperature perturbation protocols. In the first experiment, the enzyme solution, stored at 4°C with magnesium and bicarbonate ions, was warmed to the assay temperature. After temperature equilibration the carboxylase activity was assayed. The Arrhenius plot of initial rates of the carboxylase reaction is presented in Fig. 1. The results show a break point at 16°C; however, the maximum activity was low (0.1 # m o l / m i n per mg protein) [3-5,13]. In terms of the initial rate change, the calculated Arrhenius activation energy was 41.7 kcal/mol above 16°C and 67.2 kcal/mol below 16°C. In the second experiment, a heat- and CO2/Mg2+-activated enzyme preparation, which had been preincubated at 30°C for more than 1 h with Mg 2÷ and HCO3-, was brought to the respective assay temperature. After equilibration for 15 min the carboxylase activity was assayed. The Arrhenius plot of initial rates of activity was a single straight line without any breaks. The Arrhenius activation energy by this protocol was calculated to be 36.9 kcal/mol. This value is closer to the value determined above 16°C than below

16°C in the first experiment. The sedimentation velocities of the two states of the enzyme in the first temperature perturbation experiment were measured under a similar buffer solution (10 mM Tris-HC1 (pH 8.5 at 20°C) and 100 mM NaCI). The results show that the Schlieren pattern observed at either above 16°C or below 16°C is a single symmetrical, Gaussian-shaped peak (Fig. 2). The calculated sedimentation velocities corrected to 20°C in water (s20.w) were 18.8 + 0.3 and 18.1 + 0.1 S at 8.0°C and 24.4°C, respectively. The viscosity of the ribulose-l,5-bisphosphate carboxylase/oxygenase solution was anomalous; the lower the concentration of protein, the higher was the value of the reduced viscosity of the solution. The intrinsic viscosity, [7]0, was obtained by extrapolation of the experimental values of reduced viscosity between 0.3 and 5 mg/ml, and was calculated to be 28.2 d l / g at 25°C. The addition of magnesium ions to the solution increased the reduced viscosity. The values obtained with 16.1 m g / m l of protein at 25°C were 0.927 and 0.838 d l / g with and without 2 mM MgC12, respectively. The temperature response of the reduced viscosity of a 15.6 m g / m l protein solution is shown in Fig. 3. The results show that there are two distinct phases of the temperature response in terms of reduced viscosity. The transition temperature between the two phases was 15°C. The temperature

~100 c E

E

°C

I0

1 01

I

312 33

I

t

34

315 36

104/ T

K-~

Fig. 1. Arrhenius plot of initial rates ( k ) of carboxylase activity. The enzyme solution (0.98 m g / m l enzyme in 100 m M Tris-HC1 (pH 8.5 at 20°C) and 100 m M NaC1), stored at 4°C with MgCI 2 (20 mM) and N a H C O 3 (10 mM), was warmed to assay temperature; assay conditions are described in Materials and Methods. The m a x i m u m activity under these assay conditions was 0.1 ~tmol/min per mg protein.

Fig. 2. Schlieren patterns from analytical ultracentrifugation at 8.0°C (8.7 mg protein/ml) and 24.4°C (2.7 mg protein/ml). The measurements were performed in a buffer solution (10 m M Tris-HCl (pH 8.5 at 20°C), 100 m M NaC1) similar to that in Fig. 1. The calculated s20.,,, values were 18.8+0.3 and 18.1 + 0.1 S at 8.0°C and 24.4°C, respectively.

210

15 x 14

0.9

I3

p.~08 ¢

15%

07 3'3

34. 3'5 3'6 lOi/T K-~

10

20

30

40 50 rnin

611

Fig. 3 (left-hand figure). Arrhenius plot of reduced viscosity (r/red). Protein concentration was 15.6 mg/ml in a solution identical to that described in Fig. 2. Fig. 4. Time course of changes in the reduced viscosity (Ttred) when enzyme (5.3 mg/ml) stored at 4°C was transformed to 40°C. Temperature equilibration was attained within 1 min following the 4-40°C transition.

coefficients of the reduced viscosity were positive and negative above and below the transition temperature, respectively. A similar temperature response of the reduced viscosity was observed when the enzyme concentration was varied between 1 and 16 m g / m l . The enzyme stored at 4°C was transferred to 40°C, and the time course of the changes in the reduced viscosity of the solution was observed (Fig. 4). The half-relaxation time, t~/2, was about 10 min, although thermal equilibration was attained within 1 min. Discussion Badger and Collatz [14] reported that there is a break in the Arrhenius plots for the V response of both carboxylase and oxygenase activities of Atriplex leaf ribulose-l,5-bisphosphate carboxylase/oxygenase. Their preincubation conditions (at 10°C with MgC12 and NaHCO3) were similar to the first experimental protocol in the present study, and the transition temperature of the activation energy was 15°C, which is a comparable value to that (16°C) observed in the present study with the crystalline tobacco enzyme. In preliminary experiments we have also observed that the carboxylase activity of the spinach leaf enzyme showed similar biphasic features with a break at 15.6°C. These transition temperatures are essentially identical within experimental error, indicating that the dis-

continuity in the activation energy around 15°C is a common property of higher plant ribulose-l,5bisphosphate carboxylase/oxygenase. However, the tobacco enzyme displays an unusually high activation energy, 41.7 k c a l / m o l above 16°C and 67.2 k c a l / m o l below 16°C. Under identical assay conditions, the Arrhenius activation energy of the spinach enzyme was 6.2 and 29.4 kcal/mol, above and below the transition temperature. The values for the Atriplex carboxylase enzyme [14] are 15.5 and 24.8 kcal/mol, respectively. In the recent paper by Tomimatsu and Donovan [15] on a comparative study of the alfalfa, spinach and tobacco enzymes by circular dichroism and calorimetric and light-scattering methods, the tobacco enzyme displayed the smallest changes in conformation and thermal stability with changes in p H or treatment with mercurials. The significance of these unusual properties of the tobacco protein is as yet unknown. The temperature transition in activation energy of the tobacco enzyme is not accompanied by the dissociation/association of subunits of ribulose1,5-bisphosphate carboxylase/oxygenase (Fig. 2) [2,3]. However, from the temperature response of the reduced viscosity of the enzyme solution (Fig. 3), a conformational change which affects a hydrodynamic property of the protein occurs concomitantly with this transition. Chollet and Anderson [5] demonstrated that these two states of the enzyme were readily distinguished by a hydrophobic fluorescent probe and the reactivity of -SH residues. The rate of transformation between these conformers of ribulose-1,5-bisphosphate carboxylase/ oxygenase is slow. The reduced viscosity changes took more than 20 min during the temperature-shift experiment (Fig. 4), and only one activation energy of the initial rate of the heat- and CO2/Mg2+-activated carboxylase (36.9 kcal/mol) was observed. These results are consistent with the previous reports on the cold-lability of tobacco leaf r i b u l o s e - l , 5 - b i s p h o s p h a t e c a r b o x y l a s e / oxygenase [2,3,5]. Although the Tris-HC1 buffer used throughout the present experiments has a high temperature coefficient ( A p K J ° C = -0.031) [16], changes in p H of the buffer over the experimental temperatures employed are presumed not to affect the

211 p r o t e i n b e c a u s e of the o b s e r v e d l i n e a r i t y of the A r r h e n i u s plot of c a r b o x y l a s e activity i n the seco n d e x p e r i m e n t a n d the profile of p H - i n d u c e d c o n f o r m a t i o n a l c h a n g e s d e t e r m i n e d b y c i r c u l a r dic h r o i s m [ 15].

References 1 Lorimer, G.H. (1981) Annu. Rev. Plant Physiol. 32, 349-383 2 Kawashima, N., Singh, S. and Wildman, S.G. (1971) Biochem. Biophys. Res. Commun. 42, 664-668 3 Chollet, R. and Anderson, L.L. (1976) Arch. Biochem. Biophys. 176, 334-351 4 Hatch, A.L. and Jensen, R.G. (1980) Arch. Biochem. Biophys. 205, 587-594 5 Chollet, R. and Anderson, L.L. (1977) Biochim. Biophys. Acta 482, 228-240 6 Kawashima, N. and Ayabe, T. (1972) Plant Cell Physiol. 42, 523-530

7 Shirahashi, K., Hayakawa, S. and Sugiyama, T. (1978) Plant Physiol. 62, 826-830 8 Dixon, M. and Webb, E.C. (1977) Enzymes, 3rd Edn., pp. 168-169, Longman Group, London 9 Horecker, B.L., Hurwitz, J. and Wiessbach, A. (1956) J. Biol. Chem. 218, 785-794 10 Chart, P.H., Sakano, K., Singh, S. and Wildman, S.G. (1972) Science 176, 1145-1146 11 Paech, C., Pierce, J., McCurry, S.D. and Tolbert, N.E. (1978) Biochem. Biophys. Res. Commun. 83, 1084-1092 12 Kung, S.D., Chollet, R. and Marsho, T.V. (1980) Methods Enzymol. 69, 326-336 13 Bahr, J.T., Johal, S., Capel, M. and Bourque, D.P. (1981) Photosynth. Res. 2, 234-242 14 Badger, M.R. and Collatz, G.J. (1977) Carnegie Inst. Wash. Yearb. 76, 355-361 15 Tomimatsu, Y. and Donovan, J.W. (1981) Plant Physiol. 68, 808-813 16 Good, N.E., Winget, G.D., Winter, W., Connolly, T.N., Izawa, S. and Singh, R.M.M. (1966) Biochemistry 5, 467-477