Structure and function of chloroplast proteins

ARCHIVES

OF

BIOCHEMISTRY

AND

Structure VI. Further Studies

BIOPHYSICS

and

Function

of Chloroplast

on the PCMB-Treatment

T. AKAZAWA, Seikagaku

128, 646-653 (1968)

of Spinach

T. SUGIYAMA,

Seigyo Kenkyu

Shisetsu, Chikusa,

Proteins’

AND

Nagoya Nagoya,

Leaf RuDP Carboxylase

N. NAKAYAMA

University, Japan

School of Agriculture,

AND

TAKUZO Cancer Institute,

Okayama Received

University,

ODA School of Medicine,

June 25, 1968; accepted

July

Okayama,

Japan

30, 1968

The inhibitory effect of PCMB on spinach leaf RuDP carboxylase was studied by both electron microscope examination and kinetic analyses. The prior treatment of the enzyme protein with RuDP as well as NaHC03 plus Mg++ exerted a potent protective effect against the PCMB inhibition. From the results of the electron microscope studies, it was shown that the partial loss of the enzyme activity up to 50% was not definable at the ultrastructural level. But the complete loss of the enzyme activity paralleled the disorganization of the oligomeric structure of the enzyme molecule, and a prolonged contact of the enzyme protein with PCMB at high concentration produced aggregated particles to a marked degree. A simultaneous kinetic analysis of the PCMB inhibition of RuDP carboxylase showed t,hat in the lower concentration range the inhibitory effect was of the uncompetitive type. By elevating the concentration of PCMB, however, the inhibition ceases to be uncompetitive and tends to become mixed type. The overall results suggest that SH-groups are primarily engaged in the structural organization of the RuDP carboxylase molecule.

Our recent investigations have been concerned with both the structural and catalytic role of SH-groups in the wheat and spinach leaf RuDP carboxylase (14). Experimental results employing the PCMB2-titration have demonstrated the essentiality of SH-groups in holding the conformational structure of the enzyme molecule. It was shown that preliminary treatment of the enzyme protein with RuDP as well as with NaHC03 plus Mg++ exerted a protective effect against the subsequent attack by urea, SDS and

proteolytic enzyme. It has been generally accepted that RuDP carboxylase of higher plants is identical with the 17-18s protein in chloroplasts, the so-called Fraction-I protein (1, 5, 6). So far it has not been possible to dissociate the 17s protein into enzymically active smaller subunits, although there is a possibility that only a small protein of the oligomeric form is catalytically active (3, 7). In the present investigation, we have attempted to examine by electron microscope the structure of the PCMB-treated RuDP carboxylase protein which had previously been treated with either RuDP or NaHC03 plus Mg++. Our results show that the partial loss of enzyme activity is not definable at

1 This research was supported in part by a research grant of USPHS No. AM-10792.02. 2 Abbreviations. IAA, iodoacetamide; PCMB, p-chloromercuribenzoic acid; RuDP, ribulose-1,5diphosphate; SDS, sodium dodecylsulfate. 646

CHLOROPLAST

(147

PROTEINS

25 .

aB=

0.49

1 0

yci

2.0

0.5

0

FIG. 1. Inhibitory effect of PCMB on RuDP carboxylase which was treated under various conditions. Enzyme preparation (0.125 mg) was incubated with each of (a) HzO, (b) RuDP (1.75 rmoles), and (c) NaHC1403 (62.5 pmoles, 5 &i) and MgClz (12.5 pmoles) at 25” for 30 minutes (total volume 0.245 ml). During the incubation pH was maintained at 7.8. To the reaction mixture was added 5 ~1 of PCMB at their varying final concentrations as indicated in the figure. After 30 min incubation, 5.1-ml aliquot was withdrawn and added to the reaction mixture of the RuDP carboxylase assay system containing the following compositions (in pmoles): (a) Tris-HCl buffer (pH 7.8), 100; RuDP, 0.70; NaHC*403, 25.0 (2.0 &i); and MgC12, 5.0; (b) Tris-HCl buffer (pH 7.8), 100; NaHCY403; 25.0 (2.0 pCi); MgCl,, 5.0; (c) Tris-HCl buffer (pH 7.8), 100; RuDP, 0.70. Incubation was at 25” for 10 min. Measurements of WO&xation were carried out using a liquid scintillation spect,rometer.

the ultrastructural level. The overall results may indicate that SH-groups are primarily engaged in the structural organization of the enzyme molecule.3 MATERIALS

AND

METHODS

Enzyme preparation. The experimental details for preparing RuDP carboxylase from spinach leaves have appeared in our previous paper of this series (2). acfivity. This is Assay of RuDP carboxylase based upon the method reported previously (4); using the standard assay mixture (in @moles) of Tris-HCI buffer (pH 7.8), 100; RuDP, 0.70; NaHCY403, 25.0 (2.0 pCi); MgClz, 5.0; and 0.05 ml (0.05 mg or 0.13 mg protein) of the enzyme preparation in a total volume of 0.5 ml. After incubation at 25” for 10 min, 0.05 ml of glacial acetic acid was added and heated in a boiling water bath to stop 3 A part of this liminary form (8).

work

has appeared

in a pre-

the reaction. Aliquots (0.2 ml) of the heat-treated reaction mixture were then plated on steel planchets and counted in a Nuclear Chicago windowless gas-flow count,er. In other cases, the radioactivity of the fixed COZ was determined by a Packard Tri-Carb liquid scintillation spectrometer by mixing 0.2 ml of the heat-treated reaction mixture with 10 ml phosphor solution made after Bray (9). All measurements were duplicated and averaged values are presented. Electron microscopy. Samples of the enzyme protein treated with various reagents under exactly the same conditions as those used for the enzyme assay were used for t,he electron microscope studies. A drop of the reaction mixture was negatively stained with 170 phosphotungstic acid (pH 7.0) as reported previously (2). A Hitachi model 11 D High Resolution Electron Microscope was used for the ultrastructural study. Analytical ultracentrifugation. Native protein as well as t.he PCMB-treated protein samples were subjected to analytical ultracentrifugation (Beck-

FIG. 2. Electron photomicrographs of ItuDP carboxyIase molecule which was treated under various conditions. Enzyme proteins were incubated with various reagents at 25” for 30 min prior to the treatment with PCMB of various concentrations (30 min). Experimental conditions including t.he amount of enzyme protein and other reagents added was exactly the same as those explained in Fig. 1, except nonradioactive NaHC03 was used in system (c). Protein sample (j) was made contact with PCMB for 60 min. Then aliquots were withdrawn from each reaction system for preparing specimens of electron microscope examination using negative staining technique by lo/o potassium phosphotungstate (pH 7.0) as reported previousIy (2). Magnification: X 162,500. 648

CHLOROPLAST man Spinco Model E) at the Institute of Molecular Biology of Nagoya University. Experimental condition was the same reported previously (10). &age&: RuDP was prepared after the method of Horecker et al. (II), and purity was found to be i5C0 by P and pent,ose analysis. NaHC14U3 was purchased from Radiochemical Centre, Amersham, England. RESULTS

Electron microscope study. Our previous studies have shown that prior incubat,ion of RuDP carboxylase with substrat’es (RuDP or NaHC03 plus Mg++) resulted in protection of the enzyme protein against the subsequent attack by PCMB (1) and IAA (3), as well as by proteolytic hydrolysis and by urea and SDS treatment (4). We

PROTEINS

649

then examined the effect of pretreatment of the enzyme with either RuDP or KaHC03 effects of plus Mg++ against the inhibitory PCMB at various concentrations. Results shown inFig. 1 concerning the rate of enzyme activity loss vs. PCMB concent,ration were essentially in agreement w&h our previous findings [cf. Fig. 4 of (1) and (2)]. It n-ill be seen from the figure that’ a sizable number of SH-groups in the enzyme molecule are not directly involved in enzJ-me cat’alysis, and that at the point of 90% inhibition of the enzyme reaction (arrow in Fig. I), the molar ratio of SH/PCMB residues was calculated to bc approximately 1.6. This value agrees well with our previous data (4). The protective effect of RuDP was

FIG. 3. Effect of substrate (RllDP) and inhibitor (PCMB) concentrations on RuDP carboxylase. To 0.35 ml of enzyme protein (0.43 mg) was added 0.1 ml of RuDP and 0.05 ml of PCMB at, t,heir varying final concentrations as indicated in the figure. After making up the total volume to 0.5 ml, the whole mixture was incubated at 25” for 60 min. At the end of incubation, 0.2.ml aliquot was withdrawn for the enzyme assay in the reaction mixture of the following composition &moles); Tris-HCl buffer (pH 7.8), 50; NaHCIQ, 25.0 (2.0 PCi) and MgCls, 5.0 in a total volume of 0.5 ml. Radioactivity of the fixed C1hO, was determined in a Nuclear Chicago windowless gas-flow count,er by tlsing 0.2.ml aliquot, of the reactant.

650

AKAZAWA

very potent, and 50 % of the enzyme activity remained even after treating the enzyme with 2 X 10mm4 M PCMB. The molar ratio of SH/PCMB under these conditions was 0.49 (arrow in Fig. 1). A marked protective effect was also exerted by treating the enzyme with NaHC03 plus Mg++, although the pattern of their protective effect was somewhat different compared with RuDP. It can be seen that the enzyme activity of the protein molecule treated by both of NaHC03 and Mg++ was greater than the untreated native enzyme control, even in the presence of 2 X 1O-5 M PCMB. However, addition of PCMB of the higher concentrations, 1 X 1O-4 M or more, caused a gradual decline of the enzyme activity. Electron photomicrographs were taken of the protein samples incubated under exactly the same conditions as those employed for the enzyme experiments presented in Fig. 1. The letters (a-j) on the electron photomicrographs (Fig. 2) refer to the corresponding samples markeod . in Fig. 1. The cube structure, loo-120 A m diameter, consisting

b

RuDP -

0

ET AL.

of subunits, is clearly discernible in the PCMB-untreated samples (Fig. 2, a, d, g). It is notable, however, that the change in the enzyme protein which caused up to 50 % loss of activity due to PCMB-binding is not definable at the level of electron microscopy (Fig. 2, b, e, f, h, i). Only the complete loss of the enzyme activity in the protein samples without substrate treatment was accompanied by disorganization of the subunit structure (Fig. 2, c). On prolonged contact wit’h PCMB (Fig. 2, j), the enzyme preparation contained aggregated particles to a marked degree, which is in agreement with our previous study (2). Kinetic analysis of PCMB-inhibition of RuDP carboxylase yeaction. To investigate the role of SH-groups in the RuDP carboxylase molecule, in close parallel to the electron microscope studies, a kinetic study was carried out. The reaction rat’e (CY402fixation) was determined by changing the concentrations of both substrate (RuDP) and inhibitor (PCMB) molecules. In these experiments, the enzyme preparation was

0.79~10-~ M 1.11x1Ci3M 1.59~ lCj3 M 1.90~ lO-3 M 2.22d3M

8.0

4.0

PCMB

( x lT4M)

FIG. 3(b)

10.0

CHLOROPLAST

treated first with RuDP, and then with PCMB. Then an aliquot of t,he treated enzyme preparation was withdrawn for assay of RuDP carboxylase activity. Results are shown in Fig. 3. Using the double reciprocal plot of the data, it can be seen that at PCMB concentration of 1.5 X lop4 M, the inhibition was uncompetitive as indicated by the parallel lines (Fig. 3, a). However, at 1 X 1O-3 A,I, the inhibition deviated from the uncompetitive type, and tended to become mixed type. This is also supported from the results of another plot, reciprocal of the reaction rate vs. the PCMB concentrations (Fig. 3, b). The figure shows that as the substrate concentration was increased a proportionate decrease

PROTEINS

651

in inhibition occurred. The family of the parallel ‘Lcurved” lines in the plots is in contrast to the parallel, straight lines that are typical for the uncompetitive type of inhibition. Thus, it can be indicated that the more substrate bound, the more pronounced is the inhibitory effect of PCJIB on the enzyme reaction. Here again, the inhibitory effect of 1 X 1O-3 31PCMB clearly deviated from the general pattern of t,he uncompetitive type. The molar ratio of SH/PCMB at this point’ is about 0.17. Sedimentation experiments. Several lines of evidence from our previous experiments have indicated that some conformational change of the RuDP carboxylase molecule might have occurred due t’o PCMB-binding

FIG. 4. Ultracentrifugation patterns of native and PCMB-treated RuDP carboxylase. RuDP carboxylase of varying concentrations as indicated in the figure was dissolved in 0.05 M Tris-HCI buffer (pH 7.5) and was dialyzed overnight against the same buffer. Then PCMB of two different levels was added to the protein sample to attain the final concentrations as shown in the figure. Picture was taken at 21 min (control) and 19 min (PCMB) after reaching the maximum rotor speed (42,040 rpm) with a bar angle setting of 70” and temperature at 20”.

652

AKAZAWA

(1, 2). Since it was found that the kinetic properties of the reaction catalyzed by the PCMB-treated RuDP carboxylase deviated from that catalyzed by the native enzyme (4), we examined whether or not any dissociation of the protein molecule occurred by the PCMB treatment. It can be seen from the sediment,ation pattern of the enzyme protein examined by analytical ultracentrifugation (Fig. 4) that a low concentration of PCMB (6 X low4 M) cause neither discernible change in the sedimentation velocity nor the formation of aggregates; SsO being 16.7 (native enzyme) and 15.9 (PCMB) respectively. The molar ratio of SH/PCMB at this experimental condition is approximately 2.0. However, by elevating the concentration of PCMB to 1.7 X 10e3 M (SH/PCMB = approx. 0.6), the formation of aggregates is readily discernible from the sedimentation patterns, although the 175 protein is the major component. These results support those from the electron microscope st,udy. DISCUSSION

The maintainance of the rigid tertiary structure of the enzyme molecule in association with the substrate is best demonstrated by electron microscopic examination of samples from reaction mixture exactly identical wit,h those used for enzyme assays. It is not surprising to fail to detect by electron microscopy the molecular disorganization or distortion of the protein in enzyme preparations that have partially lost activity. This indicates that the loss of enzyme activity may not be ascribed to the structural breakdown of 50 % of the total enzyme molecules. Rather structural modifications not detectable by the electron microscopy might have caused alterat,ion of enzyme activity. Indeed, our previous experiments provided circumstantial evidence for the possible structural change of t.he protein molecules caused by PCMB-binding, as can be demonstrated from the enhanced proteolytic digestion (2). However, at the point of nearly complete loss of the enzyme activity, electron photomicrographs of the enzyme

ET AL.

preparation show aggregated protein structures (cf. j of Fig. 2). Usually, as in the case of our sedimentation experiments, the protein concentration is much higher than that employed in the enzyme assay system. Thus, t’he results of the sedimentation study may not be directly applicable to interpretation of the associat.ion-dissociation equilibrium of the enzyme protein. However, the maintenance of the oligomeric form of 17s protein for the RuDP carboxylase reaction as evidenced by the electron microscope study strongly supports the notion that the dissociation of t,he enzyme protein does not occur in response to the PCMB-binding. The results of the kinetic analyses portBrayed in Figure 3 indicate that PCMB at the low concentration level can combine with the enzyme protein in the presence of substrat.e, apparently not attacking the catalytic site directly. A possible interpretation would be that the dissociation constant (KEI) of the enzyme inhibitor complex (EI) is indefinitely large and that the latter is unlikely to be formed in the reaction process. However, by elevating the concentration, PCMB appears to attack SH-groups which are probably located close to the substratebinding site, although further detailed study is needed to substantiate this point,. Our previous st.udies have not given any conclusive evidence concerning the catalytic role of SH-groups in the carboxylation reaction (3). It is likely from the results of the present kinetic analyses that SH-groups occupy a very important role in the structural organization of t.he enzyme molecule. REFERENCES 1. SUGIYAMA, T. AND AKAZAWA, T., J. Biochem. 62, 474 (1967). 2. SUGIYAMA, T., NAKAYBMA, N., OGAWA, hf., AKAZAWA, T., AND ODA, T., Arch. Biochem. Biophys. 126, 98 (1968). 3. SUGIYAMA, T., AKAZAWA, T., NAKAYAMA, N., ASD TANAKA, Y., Arch. Biochem. Biophys. 136, 107 (1968). 4. SUGIYAMA, T., NAKAYAMA, N., TANAKA, Y.,

CHLOROPLAST AND AKAZ~I~~, T., Arch. Biochem. Biophys. 126, 181 (1968). 5. TROIVN, I'. W., Riochemisfry 4, 908 (1965). 6. RIDLEY, S.RI.,THOKNRER, J. P., AND BAILEY, J. I,., Biochim. Biophys. Ada 140, 62 (1967). 7. HGELKORN, R., FERNANDEZ-MORAN, H., KIERAS, F. J., AND VAN BRUGGEN, E. F. J., Science 150, 1598 (1965).

653

PROTEIN8

8. SUGIYAMA, T., AKAZAJVA, T., AND NABAYAMA, N., ilrch.

Biochem.

Biophys.

121, 522 (1967).

9. BRAY, G. A., Anal. Biochem. 1, 279 (1960). 10. SUGIYAMA, T., NAKAYAMA, N., AND AKAZAI\-A, T., Arch. Biochem. Biophys. In press. (19G8). 11. HOKECKER, B. L., HURI~VITZ, J., AND WEISSUACH, A., J. Biol. Chew 218, 785 (1956).