- Email: [email protected]
oxygenase from Rhodospirillum rubrum
ARCHIVES Vol.
OF BIOCHEMISTRY
219,
No.
2, December,
AND pp.
BIOPHYSICS
422-437,
1982
Purification and Sequencing of Cyanogen Bromide Ribulosebisphosphate Carboxylase/Oxygenase Rhodospirillum rubrum’ FRED Biology
Division,
C. HARTMAN,’ CLAUDE MARK I. DONNELLY,4
D. STRINGER, AND BASSAM
Fragments from
JOHN FRAIJ5
OMNAAS,”
Oak Ridge National Laboratory and the University of Tennessee-Oak School of Biomedical Sciences, Oak Ridge, Tennessee 37830 Received
June
23,
from
Ridge
Graduate
1982
As a part of the goal to determine the total sequence of Rhodospirillum rubrum ribulosebisphosphate carboxylase/oxygenase, the cyanogen bromide fragments were fractionated and sequenced (or partially sequenced). Twelve of the anticipated 14 peptides were obtained in highly purified form. The other two peptides were located, respectively, within a trytophanyl cleavage product (which overlapped with four CNBr fragments) and within an active-site peptide characterized earlier (which overlapped with three CNBr fragments). These overlaps coupled with amino and carboxyl terminal sequence information of the intact subunit and the availability of the sequence of the corresponding enzyme from higher plants permitted alignment of all fragments. Eight CNBr peptides were sequenced completely; four of the CNBr peptides consisted of more than 80 residues and were only partially sequenced as permitted by direct Edman degradation. Of the approximate 47.5 residues per subunit, 339 were placed in sequence. The lack of extensive conservation of primary structure between R. rubrum and higher plant carboxylases permits the tentative identifications of those regions likely to be functionally important.
As the enzyme that catalyzes the initial step in photosynthetic CO, fixation and in photorespiration (see Ref. (1) for review),
ribulose-PZ6 carboxylase/oxygenase (EC 7.1.1.39) plays a pivotal role in plant growth and productivity and thus has become the subject of intense structural and mechanistic studies. Because of conservation of essential molecular features during evolution, comparative sequence analyses of functionally analogous enzymes provide a useful approach to discern regions of primary structure essential either to catalysis, regulation, or integrity of
’ The U. S. Government’s right to retain a nonexclusive royalty-free license in and to the copyright covering this paper, for governmental purposes, is acknowledged. Research sponsored jointly by the Science and Education Administration of the U. S. Department of Agriculture under Grant 78-59-2472-0-1161-1 from the Competitive Research Grants Office and by the Office of Health and Environmental Research, U. S. Department of Energy, under Contract W-7405.eng-26 with the Union Carbide Corporation. * To whom all correspondence should he addressed. 3 Postdoctoral investigator supported by Suhcontract 3322 from the Biology Division of the Oak Ridge National Laboratory to the University of Tennessee. 4 Postdoctoral investigator supported by NSF Grant PCM-7908990. ’ Postdoctoral investigator supported by the U. S. 0003.9861/82/140422-16$02.00/O Copyright All rights
0 1982 by Academic Press, Inc. of reproduction in any form reserved
Department of Agriculture grant through Suhcontract, 3322 from the Biology Division of the Oak Ridge National Laboratory to the University of Tennessee. 6 Abbreviations used: Rihulose-P,, D-rihulose 1,5bisphosphate; Na-dodecyl-SO,, sodium dodecyl sulfate; DMAA, N,N-dimethyl-N-allylamine; and Quadrol, N,N,N’,N’-tetrakis(2-hydroxypropyl)-ethylenediamine. 422
SEQUENCE
OF
RIBULOSE-P,
three-dimensional structure. Although complete primary structures of the large subunits of ribulose-P, carboxylase from both maize and spinach have been deduced by cloning and sequencing their respective genes (2, 3), little is learned concerning which residues are critical to function, because these two carboxylases exhibit 90% homology. In contrast, comparisons between the carboxylases from higher plants and the purple non-sulfur bacterium Rhodospirillum rubrum appear especially appropriate as these organisms are evolutionarily quite distant (4) and their respective carboxylases differ markedly in both primary and quaternary structure. For example, ribulose-P2 carboxylase from higher plants consists of eight large (-53,000 daltons) and eight small (-14,000 daltons) subunits (2, 5, 6), whereas the enzyme from the purple nonsulfur bacterium Rhodospirillum rubrum is a simple dimer of large subunits (7, 8). Total sequence homology appears to be no greater than 20% (9, 10); thus, stretches of homology that are found are likely to be crucial to function. We previously determined the sequence of cysteine-containing peptides from tryptic digests of R. rubrum ribulose-P2 carboxylase in order to ascertain if any cysteinyl residues were species invariant; because homologies were not observed, we suggested that sulfhydryl groups are not essential to catalysis (10). We now report purification and sequencing of cyanogen bromide fragments, which account for virtually the entire polypeptide chain; about 70% of the total sequence has been obtained. Two lysyl residues previously implicated at the active site are observed to be located in highly conserved regions. EXPERIMENTAL
PROCEDURES
Materials. Ribulose-P2 carboxylase/oxygenase from R. rubrum was purified to homogeneity according to a published procedure (8). Cyanogen bromide and polybrene were obtained from Aldrich Chemical Company. Other chemicals and vendors were iodosobenzoic acid from Pierce, Carboxypeptidase Y from Sigma, ultrapure urea from Schwarz/Mann, pepstatin from United States Biochemical, and [3H]iodoacetic acid from New England Nuclear. Chemicals, buffers, and reagents required for amino acid analyses and for
CARBOXYLASE
423
automated Edman degradations were purchased from either Pierce Chemical Company or Beckman Instruments, Inc. Ingredients for Na-dodecyl-SO,-polyacrylamide gel electrophoresis were products of Bethesda Research Laboratories. General methods. Preceding cleavage with either cyanogen bromide or iodosobenzoic acid, the carboxylase was carboxymethylated with [3H]iodoacetic acid (sp act = 1.7. lo6 cpm/wmol) as described earlier (10). The alkylated protein had a sp act of 9.5.10s cpm/ pmol representing an incorporation of 5.6 mol of carboxymethyl groups per mole of subunit. Urea was deionized by batchwise treatment with Bio-Rad AC, 501-X8. A Packard 3255 liquid scintillation spectrometer was used to measure radioactivity. Samples were diluted to 0.5 ml with water and then dissolved in 10 ml of ACS counting solution (Amersham Corporation). During column chromatography, peptides were detected by continuous monitoring of effluents’ absorbancies at 206,215, or 280 nm with a LKB Uvicord III or a Gilson Holochrome. Peptides were dialyzed in Spectrapor 2000 membrane tubing (Spectrum Medical Industries). Electrophoresis. Na-dodecyl-SO,-polyacrylamide slab gel electrophoresis was carried out according to procedures of either Laemmli (11) or Shapiro et al. (12). For the monitoring of peptides during their purification, the Laemmli (11) system was used with a microslab apparatus (13) available commercially from Idea Scientific, Corvallis, Oregon. The acrylamide concentration was 15%, and the gel dimensions were 100 mm in width X 90 mm in height X 0.5 mm in thickness. A slab gel apparatus from Bethesda Research Laboratories (Model V16) was used according to the manufacturer’s instructions [slight modifications of those of Shapiro et al. (12)] to estimate molecular weights of peptides. The acrylamide concentration was 15%, and the gel dimensions were 16.8 cm in width X 16 cm in height X 3 mm in thickness, In all cases, peptides were visualized with Coomassie blue (0.1%) in methanol:water:acetic acid (5:5:1); the same solvent was used for destaining. Amino acid analyses. Total acid hydrolysis of peptides were achieved in evacuated (-50 pm Hg) sealed tubes with 6 N HCl/O.Ol M 2-mercaptoethanol at 110°C for 21 h. Hydrolysates were dried on a Speed Vat Concentrator (Savant Instruments Inc.) and subjected to chromatography on a Beckman 121M amino acid analyzer using Beckman’s “3-hour-single-column system.” The analyzer was hard wired to a PDP data acquisition system. Computer programs used in data collection and analysis were provided by S. S. Stevens and J. T. Holderman of the Oak Ridge National Laboratory. Separate samples were hydrolyzed with 3 N mercaptoethanesulfonic acid for the determination of tryptophan content (14); hydrolysates were brought to pH 2.0 with 3 N NaOH and diluted lo-fold with H,O prior to analysis.
424
HARTMAN
Digestion with cyanogen bromide (15). To a solution of carboxymethylated ribulose-P, carboxylase (430 mg, 7.7 pmol of subunit) in 40 ml of 70% (v/v) formic acid was added 400 mg (3.8 mmol) of CNBr dissolved in 2 ml of 70% formic acid. The digestion was allowed to proceed in the dark at room temperature for 20 h at which time the reaction was terminated by the addition of H,O (600 ml) and lyophilization of the diluted solution to dryness. The sample was redissolved in 60 ml of 0.1 N acetic acid, subdivided into aliquots containing 40 mg of digested protein, lyophilized, and then stored at -80°C. Digestion with iodosobenzoic acid (16-18). Iodosobenzoic acid (170 mg) was added to a solution of carboxymethylated ribulose-Py carboxylase (86 mg, 1.54 pmol) consisting of 80% (v/v) acetic acid (10 ml), 8 M guanidine hydrochloride (1.8 ml), and thiodiglycol (45 ~1). After 5 min in the dark at room temperature, the reaction mixture was quenched by the addition of 2-mercaptoethanol (660 ~1) and desalted on a 2.5 X 19-cm column of Bio-Gel P-2. The excluded peptide region was dried by lyophilization. Digestion with carboxypeptidase Y (19). Commercial enzyme (10 mg) was partially purified by gel filtration at 4°C on a 2 X 82.cm column of Bio-Gel P-100 equilibrated with 0.01 M Na2HP0,/0.1 M NaCl/l mM EDTA (pH 7.0) (19). To the peak fraction (2 ml, 14 @M in carboxypeptidase) was added 20 ~1 of pepstatin (50 mg/ml in dimethyl sulfoxide). Digestion of peptides with carboxypeptidase Y was carried out at room temperature in 0.1 M pyridine acetate (pH 5.5) containing norleucine as internal standard; other specific conditions are provided in figure legends. Periodically, loo-p1 aliquots of the digestion mixtures were diluted into 2 ml of boiling water; the diluted aliquots were centrifuged to remove insoluble material, and the supernatants were lyophilized to dryness, dissolved in 300 ~1 of 0.1 M sodium citrate (pH 2.2), and then subjected to amino acid analyses. All calculations were normalized to the norleucine internal standard. Sequence analyses. Purified peptides (36-190 nmol) were subjected to automated Edman degradations with a Beckman 890C sequencer, the vacuum system of which was modified according to Bhown et al. (20). A liquid nitrogen cold trap was inserted between the low vacuum pump and the vacuum manifold. Polybrene (2 mg) was added to the peptide to reduce its extraction from the reaction cup (21). Both dimethylallylamine and Quadrol buffer systems were employed; Beckman’s peptide program 102974 was used with the former. The program used with the Quadrol buffer was that of Bhown et al. (20) with several modifications. The buffer concentration was reduced from 0.5 to 0.1 M, the drying step after delivery of phenyl isothiocyanate was increased from 20 to 60 s, and the coupling steps and drying steps after delivery of benzene/ethyl acetate were from Beckman’s peptide program 030176. Generally, half of the fraction from each
ET
AL.
cycle was hydrolyzed in base for quantitation as free amino acid on the amino acid analyzer (22). Threonine and arginine appear as ol-aminobutyric acid and ornithine, respectively, in base hydrolysates. The other half of each fraction was converted to the phenylthiohydantoin for identification by high-performance liquid chromatography (Laboratory Data Control); this provided confirmation of the identifications made by amino acid analyses and distinguished aspartic acid and glutamic acid from the corresponding amides. Tentative identifications of PTH-serine based on HPLC and the presence of a degradation product in the base hydrolysates that coelutes with cysteic acid on the amino acid analyzer were confirmed by carrying out acid hydrolysis on a portion of the PTH derivative thereby converting serine to alanine (23). For those peptides that were radioactive, 10% of each fraction from the sequencer was counted so as to identify carboxymethylcysteine; the remainder of the fraction was then processed as already described. RESULTS
Purification of CNBr fragments. Initial fractionation of CNBr fragments was achieved by ion-exchange chromatography on carboxymethyl cellulose at pH 4.0 in the presence of 8 M urea (Fig. 1). Each peak was inspected by Na-dodecyl-SO,polyacrylamide gel electrophoresis; all of the components that eluted before initiation of the salt gradient were small peptides as they could not be visualized by staining the gels with Coomassie blue. Peaks C8, C9, ClO, Cll, C17, and Cl8 represented complex mixtures of fragments larger than 14,000 daltons, which were assumed to be incomplete digestion products and were thus not processed further. The four major large fragments in the CNBr digest (see Fig. 2, lanes 2 and 7) were found in the regions denoted C12-C13, C14-C15, C16, and C19. Fractions from these regions were separately pooled, and the four pools were dialyzed against 0.5% (v/v) acetic acid and then lyophilized to dryness. The lyophilized samples were dissolved in 8 M urea buffered at pH 5.0 with 0.01 M ammonium acetate and rechromatographed on carboxymethyl cellulose (Fig. 3). The center fractions of the four major peaks were pooled as indicated in the figure, dialyzed against 0.5% acetic acid, and lyophilized. Each of the four large peptides (denoted C12, C14, C16, and C19) thus
SEQUENCE
OF
RIBULOSE-Ps
425
CARBOXYLASE
0 0
b
Ii0
150*0
2fil4oto
io
do
VOLUME (ml)
FIG. 1. Ion-exchange chromatography of a cyanogen bromide digest of “H-carboxymethylated ribulose-P, carboxylase on Whatman CM-52 carboxymethyl cellulose. The sample (46 mg) in 2.0 ml of 0.01 M ammonium formate/ M urea (pH 4.0) was applied to the column (1 X 50 cm) equilibrated with the same ammonium formate/urea buffer. The column was eluted first with 150 ml of equilibration buffer followed by two successive 400.ml linear gradients (O-0.1 M NaCl and 0.1-0.2 M NaCl) prepared with the same urea-containing equilibration buffer.
obtained appeared highly pure by gel electrophoresis (Fig. 2). Based on mobilities relative to those of the standards also
12345678
FIG. 2. Na-dodecyl-SO,-polyacrylamide gel electrophoresis of the total cyanogen bromide digest of carboxymethylated ribulose-P, carboxylase (lanes 2 and 7) and the purified large fragments Cl2 (lane 3), Cl9 (lane 4), Cl4 (lane 5), and Cl6 (lane 6). A standard from Pierce containing soybean trypsin inhibitor (21.5K). cytochrome c (12.5K), aprotinin (6.5K), and insulin B chain (3.4K) is shown in lanes 1 and 8. See Experimental Procedures for further details.
shown in Fig. 2, peptides C12, C14, C16, and Cl9 have molecular weights of ll.lK, 8.2K, 7.8K, and 9.4K, respectively (Fig. 4). The smaller peptides represented by peaks C2 through C7 (Fig. 1) were all purified by essentially the same proceduresfirst, gel filtration on Sephadex G-25, and second, when necessary, ion-exchange chromatography on DEAE-cellulose. In the case of C3, gel filtration on a G-25 column in the presence of 6 M urea was necessary to remove a contaminating peptide whose amino acid composition was the same as C5; C3 was then freed of urea by desalting on a G-25 column equilibrated with 0.01 M NH,HC03 (pH 8.0). Lyophilization served to concentrate and desalt all peptides obtained from DEAE and G25 columns when NH,HCO, (in the absence of urea) was the buffer. Peptide material could not be detected in peak Cl (Fig. 1) by amino acid analysis suggesting that this peak represents a change in refractive index associated with the solvent front. Purifications of peptides C2, C3, C4a, C4b, C5, C6a, C6b, and C7 are shown in Figs. 5 and 6. Gel filtration of peaks C2 and C3 combined yielded C2 as a pure peptide (Fig. 5A). Peptide C3 was obtained from the pool of C3 plus C5 (Fig. 5A) by gel filtration in the presence of urea which removed peptide C5 (Fig. 5B). Amino acid analyses of the initial two peaks in Fig. 5A
426
HARTMAN
0.2
64
ET
AL.
a4
0.07
_.^
Cl4
0.07
0.6-
I 0.20
CG
i ;
1
/’ 27
7
0.16
/ /’ 169
243
400
27
61
135
169
243
400
FIG. 3. Rechromatography of the four large cyanogen bromide fragments (C12, C14, C16, and C19) on a column (1 X 50 cm) of Whatman CM-52 carboxymethyl cellulose equilibrated with 0.01 M ammonium acetate/8 M urea (pH 5.0). In all four cases the column was eluted with a 400-ml linear gradient of sodium chloride dissolved in the urea-containing equilibration buffer. The final sodium chloride concentration was as indicated in each panel.
suggested that these corresponded to peptides C6b and C4a in impure forms. Chromatography of peptide C4 (obtained from gel filtration, Fig. 4C) on DEAE-cellulose resolved peptides C4a and C4b (Fig. 6A). Peak C5 chromatographed as one major component on both G-25 and DEAE-cellulose (Fig. 5D and Fig. 6B). Peak C6 was resolved into peptides C6a and C6b by gel filtration (Fig. 5E) and each of these was purified further on DEAE-cellulose (Figs. 6C and D). A single peptide was obtained from peak C7 (Figs. 5F and 6E). Purification of two trytophanyl cleavage products. The lyophilized digest of the car-
boxylase (86 mg) that had been treated with o-iodosobenzoic acid (see Experimental Procedures) was extracted with 10 ml of 10 mM acetic acid, and the insoluble material was removed by low-speed centrifugation. The supernatant was brought
to 60 mM ammonium bicarbonate thereby resulting in the precipitation of additional
20.~ a
,o-1 ‘& F * 5-
a\ 4
31 4
5
6 7 MIGRATION
e 9 DISTANCE (cm)
IO
4
I1
FIG. 4. Determination of molecular weights of the large cyanogen bromide fragments based on the gel shown in Fig. 2. The open circles represent the standard proteins: soybean trypsin inhibitor (21.5K), cytochrome c (12.5K), aprotinin (6.5K), and insulin B chain (3.4K).
SEQUENCE
OF
RIBULOSE-P2
,6 , 1
I-: >
/60 I I 30 T s d 2 b x
427
CARBOXYLASE
the sample was subjected to gel filtration on Sephadex G-50 (Fig. 7B). The second peak (12) was purified by cation-exchange chromatography on SP-Sephadex (Fig. 7C). Amino acid compositions of purified peptides. Results from amino acid analyses of the purified peptides are provided in Table I along with the percentage recoveries of each peptide, estimates of total number of residues and molecular weights, and specific radioactivities of those five peptides containing carboxymethyl cysteine. The isolation of five labeled CNBr peptides with similar specific activities, about onefifth that of the intact polypeptide (see Experimental Procedures), is consistent with the presence of only one residue of carboxymethyl cysteine in each and ac-
E
i
‘2
A
A
c4a C4b
20 IO
20 10
140
180 VOLUME
220
i
!6(1
0.4 Am
(ml1
FIG. 5. Gel filtration of peaks C2 + C3 (A and B), C4 (C), C5 (D), C6 (E), and C7 (F) that were obtained from the CM-cellulose column (see Fig. 1). The column (1.7 x 220 cm) consisted of Sephadex G-25 equilibrated and eluted with 0.01 M NH,HCOB (pH 8.0). For the resolution of peptides C5 and C3 (B) the NH,HCO? buffer contained 6 M urea.
0.2 P
8 ‘, 5
/’ .0’ E
peptides which were again removed by centrifugation. The supernatant, which contained peptides soluble in 60 mM ammonium bicarbonate, was fractionated on Sephadex G-25 (Fig. 7A). Rechromatography of peptide 11 on the same column yielded this material in a highly pure form. Fractions from the excluded region (Fig. 7A) were lyophilized; the residue was dissolved in 0.5 ml of 8 M guanidine hydrochloride, and following the addition of HZ0 (1 ml)
c7 :\
04 0.2
,’
,’ i c, _I,.-
,’
,’
,’
0.4
- ’ ’
0.2
m 100
200 VOLUME
300
400
irnl)
FIN;. 6. Ion-exchange chromatography of peptides after their subjection to gel filtration (see Fig. 5). The column (1 X 25 cm) consisted of Whatman DE-52 DEAE-cellulose equilibrated with 0.01 M NH,HCOB (pH 8.0) and eluted with 400.ml linear gradients of NH,HCO, (pH 8.0) as specified by the ordinates.
428
HARTMAN
counts for the five cysteinyl residues per subunit (10). In the smaller peptides, the absence of certain amino acids and the approximate integral ratios of residues indicate a high degree of purity. Amino acid analyses of the four large fragments (Cl2, C14, C16, and C19) are not informative concerning degree of purity; however, as already indicated, these peptides were homogenous asjudged by acrylamide gel electrophoresis (Fig. 2). The molecular weights of peptides C14, C16, and Cl9 calculated from amino acid analyses were somewhat greater than those determined by gel electrophoresis. The amino acid composition of peptide C7 was identical to that of peptide C6b except the former contained one residue of methionine. Carboxyl terminal analyses of intact subunit, peptide C16, and peptide Il. Because of the absence of homoserine in peptide C16, it was assumed to be the carboxyl terminal fragment. Analyses of this fragment and the intact carboxymethylated subunit by digestion with carboxypeptidase Y were consistent with a carboxyl terminal sequence of Ala-Leu-Pro-Ala (Figs. 8A, B). Although leucine and proline were released at very similar rates, proline is placed as the penultimate residue on the basis of alanine, in low yield, as the only product of digestion with carboxypeptidase A (data not shown). The excess leucine observed during digestion of carboxymethylated subunit with carboxypeptidase Y is likely a consequence of a leucine-specific endopeptidase which contaminates commercial preparations (19, 24). Greater than stoichiometric amounts of leucine and many other amino acids were released when peptide Cl6 was digested with carboxypeptidase Y that had not been subjected to gel filtration followed by treatment with the endopeptidase inhibitor pepstatin as described under Experimental Procedures. Furthermore, without these precautions extensive degradation of Cl6 into multiple discrete peptides was observed by Na-dodecyl-Sod-gel electrophoresis. Since peptide 11, obtained by fragmentation of the whole subunit with iodosobenzoate, lacked the characteristic absorbance of oxidized indole (25),
ET AL.
FIG. 7. Purification of two tryptophanyl cleavage peptides from ribulose-Pp carboxylase. (A) The mixture of peptides that were soluble in ammonium bicarbonate (see Results) was fractionated on a 1.5 X 230.cm column of Sephadex G-25 equilibrated with 0.01 M NH,HCOs (pH 8.0). Peptide II was rechromatographed on the same column (profile not shown). (B) Peptide I2 from (A) was subjected to gel filtration on a 1.5 X 215cm column of Sephadex G-50 equilibrated with 0.01 M NH,HCOs (pH 8.0). (C) Peptide I2 from (B), after lyophilization, was redissolved in 2.0 ml of 6 M urea/32 mM H,PO, (pH 3.0) and subjected to cation-exchange chromatography on a 1 X 20.cm column of Sulfopropyl Sephadex C-25. The column was equilibrated with 6 M urea/32 mM H,PO, (pH 3.0) and eluted with a 400.ml linear sodium chloride gradient (O-1.3 M) prepared with the same ureacontaining buffer.
it too was tentatively identified as a carboxyl-terminal segment. Digestion with carboxypeptidase Y confirmed the -LeuPro-Ala-COOH sequence (Fig. 8C). Sequence analyses of purified peptides. Automatic Edman degradations were carried out on the purified peptides (Tables I-XIV in miniprint supplement); the lack of a second (or multiple) residue at cycle one in quantities greater than 5% of the residue identified as the amino terminus verified the high degree of purity of these peptides. Although the results for only a single analysis of each peptide are shown, the established sequences (or partial se-
2.2 (2)
0.1
GUY Ala
MI
Recovery
Calcd.
residues
Total
A%
TOP
LYS
His
TY~ Phe
28
24
50
11
1077
9
996
6
0.2
4.3 (2)
-
2.3 (1)
0.2
2.3 (1)
2.1 (1)
0.1
623
1.1 (1)
2.1 (2)
1.2 (1)
Ile
Leu
-
Met
(%)
1.1 (1)
1.2 (1)
Glu Pro
0.1
2.0 (1)
0.1
0.1
Val
3.9 (2)
2.0 (2)
0.1
Ser
1.0 (1) -
0.1
1.6 (1)
2.2 (1)
2.1 (2)
0.8 (1)
C4a
1.0 (1)
c3
Cys (Cm) Asp Thr
c2
Homoserine
Amino acid
20
1328
12
1.6 (1)
1.4 (1)
8 912 56
18 34
_
1.1 (1)
1.0 (1)
2074
1.4 (1)
1.6 (1)
1.4 (1)
1.4 (1)
1.6 (1)
0.1
20
3240
29
1.1 (1)
1.2 (1)
1.0 (1)
2.0 (2)
4.2 (4)
2.1 (2)
0.2
2.1 (2)
3.2 (3)
1.0 (1)
1.4 (1) 1.0 (1)
2.3 (2)
4.6 (4)
2.1 (2)
3.3 (3)
1.1 (1)
0.8 (1)
C6b
1.0 (1)
1.0 (1)
0.9 (1)
0.9 (1)
C6a
3.8 (3)
1.4 (1)
5.0 (4)
1.3 (1)
1.3 (1)
1.4 (1)
1.2 (1)
C5
9
3371
30
1.1 (1)
1.3 (1)
1.0 (1)
-
2.0 (2)
4.2 (4)
2.1 (2)
0.8 (1)
2.1 (2)
3.2 (3)
2.3 (2)
4.6 (4)
2.1 (2)
6.0 (6)
4.7 (5)
14 25
1401
29
11.8K
110
4.6 (5)
6.2 (6)
1.9 (1)
1.9 (2)
1.8 (1)
0.1
2.0 (2)
6.0 (6)
-
8.4 (8) 15.1 (15)
12.4 (12) 13.4 (13)
35
lO.OK
92
51
9.5K
88
29
10.8K
98
8.0 (8)
1.8 (2)
6.8 (7)
3.2 (3)
2.2 (2) 2.7 (3)
5.2 (5) 4.0 (4)
5.0 (5)
6.0 (6)
5.0 (5)
5.3 (5) 4.0 (4)
7.0 (7) 2.0 (2)
3.6 (4)
2.8 (3)
7.8 (7)
3.1 (3)
6.4 (6)
4.0 (4)
8.6 (9)
4.0 (4)
6.9 (7)
8.0 (8)
1.1 (1)
0.9 (1)
Cl9
8.4 (8)
3.4 (3)
2.0 (2)
9.2 (9)
Cl6
2.0 (2) 1.8 (2)
9.2 (9)
5.2 (5)
2.0 (1)
4.4 (4)
7.0 (7)
10.0 (10)
3.3 (3)
11.3 (11)
17.0 (17)
9.4 (9)
8.3 (8)
3.4 (4) 9.4 (9)
7.2 (7)
2.8 (3)
2.0 (2)
10.4 (IO)
0.9 (1)
0.7 (1)
Cl4
8.6 (9)
3.1 (3)
11.3 (11)
12.4 (12)
1.3 (1)
0.9 (1)
Cl2
2.0 (1)
-
~
3.9 (2)
5.8 (3)
2.0 (1)
1.9 (1)
3.8 (2)
0.1
3.3 (3) -
-
1.8 (1)
Cl3
1.1 (1)
1.0 (1)
C7
Peptide (nmol found and number of residues in parentheses)
0.1
1.4 (1)
0.02
3.1 (2)
1.6 (1)
2.9 (2) -
2.9 (2)
1.5 (1)
1.4 (1)
C4b
I
AMINO ACID COMPOSITIONSOF PURIFIED FRAGMENTS
TABLE
32
1584
15
2.5 (2)
1.3 (1)
-
~
2.5 (2)
-
1.4 (1)
3.8 (3)
1.5 (1)
1.3 (1)
1.4 (1)
1.4 (1)
1.3 (1)
1.4 (1)
-
I1
8
5449
56
1.2 (2)
0.6 (1)
0.6 (1)
1.7 (3)
2.2 (4)
2.3 (4)
1.6 (3)
1.0 (2)
4.6 (8)
8.1 (13)
1.9 (3)
1.1 (2)
1.0 (2)
1.3 (2)
3.2 (5)
0.4 (1)
I2
HARTMAN
430
ET
TABLE SEQUENCE
AL.
II
OF PURIFIED
FRAGMENTS’
Peptide c2
Gly-Asp-Asp-Glu-G;y-Hse
c3
Ile-Ala-Ser-Phe-Leu-Thr-Leu-Thr-Hse
C4a
Lys-Ala-Cys-Thr-P:o-Ile-Ile-Ser-Gly-G!y-Hse
C4b
Glu-Gly-Glu-Ser-&-Asp-Arg-AlaIle-;!a-Tyr-Hse
c5
Leu~Thr~Gln-Asp-G;u-Ala-Glu-Gly-Pro-~~e-Ty~-A~g-Gl”-Se~-~~-Gly-Gly-Hse
C6a
Gly-Asp-Val-Glu-Tir-
C6b
Asp-Gln-Ser-Ser-A~g-Tyr-Val-Asn-Le~-~~a-Le~-Lys-Gl~-Gl~-~~p-Le~-Ile-Ala-Gly-~~y25 Glu-His-Val-Leu-Cys-Ala-Tyr-Ile-Hse
Cl3
Ala-Arg-Leu-Gln-G~y-Ala-Ser-Gly-Ile-~~s-Th~-Gly-Th~-Hse
Cl2
Lys-Pro-Lys-Ala-G~y-TyrGly-Tyr-Val-~~a-Th~-Ala-Ala-His~~~e-Ala-Ala-Gl~-Se~-~~~25 3” Thr~Gly~Thr-Asp-Val-Glu-Val-Cys-Thr-Thr-Asp-Asp-Phe-Th~~~~g-Gly-Val-Asx-Ala-L~~ 1.5 -Val-Tyr-Glx-Val-Asx-Gly-Ala-----
Cl4
His-Asp-Phe-Tyr-V~l-Pro-Glu-Ala-Ty~-~~g-Ala-Le”-Phe-Asp-~~y-P~o-Se*-Val-Asn-~25 30 Ser-Ala-Leu-Trp-Lys-Val-Leu-Gly-Arg-Pro-Gl~-Val-Asp-Gly-Gly-Le~-Val-Val-Gly-~~~45 Ile-Ile-Lys-Pro-Lys-Leu-Gly-Leu-Arg-~~o-----
Cl6
Pro-Gly-Phe-Phe-G~n-Asn-Leu-GlyAsn-~~a-Asn-Val-Ile-Le~-~~~-Ala-Gly-Gly-Gly-~~a-
Ala-Lys-Hse
35
Phe-Gly-His-Ile-~~x-Gly-Pro-Val-Ala-~~y-Ala-A~g-Se~-Le~-~y-Glx-Ala-T~-Glx------~ (COOH-terminus Cl9
fragment)
Arg-Arg-Ala-Gln-A~p-Glu-Thr-Gly-Glu-~~a-Lys-Leu-Phe-Ser-A$-Asn-Ile-Thr-Ala~~~pAsp-Pro-Phe-Glu-~-Ile-Ala-Arg-Gly-~~x-Tyr-Val-Leu-Le~-~~~-Phe-Phe-Gly-AsxA”$--------
11
Arg-Lys-Ala-Leu-C;;y-Val-Glu-Asp-Th~-~~g-Se*-Ala-Leu-Pro-A~a
12
Gly-Gly-Met-Lys-A~a-Cys-Thr-Pro-Ile-~~-Se~-Gly-Gly-Met-d~n-Ala-Leu-Arg-Met-~~o-
/
Gly-Phe-Phe-Gln-A~n-Leu-Gly-Asn-Ala-A~n-Val----’ Cysteinyl
residues
are carboxymethylated;
Hse,
homoserine.
quences) (Table II) were confirmed by duplicate degradations. Peptide C7 differed from peptide C6b only in having a methionine added to the amino terminus (data not shown). This sequence is identical to that determined earlier by Edman degradation of the intact subunit (8); thus, C6b and C7 are both derived from the amino end of the subunit and the latter is a consequence of incomplete cleavage of the amino terminal methionyl residue. Alignment of fragments. A comparison of the partial sequence of the R. rubrum
carboxylase and the total sequence of the spinach enzyme large subunit, which was determined earlier (3), is illustrated in Fig. 9. The alignments of the four large CNBr fragments are straightforward. The amino terminal fragment C6b is contiguous with fragment Cl2 based on obvious homology from positions 53 to 72;7 furthermore, by 7 Because of deletions and insertions, the two carboxylases are not exactly the same length; however, for simplicity the residue numbers of the spinach carboxylase are applied to the R. rubrum carboxylase.
SEQUENCE
OF
RIBULOSE-Ps
431
CARBOXYLASE
from the R. rubrum enzyme containing Met-335 (27, 28), which is selectively alkylated with an affinity label, provides an overlap for C13, for a fragment with the sequence Gly-Phe-Gly-Lys-Met (which was not isolated from the CNBr digest), and for C4b. Fragment 12, obtained by digestion of the enzyme with iodosobenzoic acid, overlaps four peptides: C5, C4a, a fragment with the sequence Asn-AlaLeu-Arg-Met (which was not isolated from the CNBr digest), and C16. The peptides in Fig. 9 designated with the letter “T” represent carboxymethyl cysteinylcontaining peptides from tryptic digests characterized previously (10). Placements of peptides C2, C3, C6a, T4B, and T4C are based on apparent homologies and/or seemingly proper lengths and therefore must be considered as tentative. r
10
xl TIME (mm)
30
FIG. 8. Digestion of peptide Cl6 (A), intact carboxymethylated ribulose-P, carboxylase (B), and peptide I1 (C) with carboxypeptidase Y. Digestions were carried out at room temperature in 0.1 M pyridine acetate (pH 5.5); pepstatin, dimethyl sulfoxide, and norleucine were also present at final concentrations of 500, 800, and 200 pM, respectively. In the cases of peptide Cl6 and intact subunit, the pyridine acetate buffer contained 5 M urea as a solubilizer. Final concentrations of peptides and carboxypeptidase Y in the digestion mixtures were as follows: 89 FM Cl6 with 0.64 pM carboxypeptidase, 82 FM whole subunit with 0.55 WM carboxypeptidase, and 79 pM I1 with 4.4 pM carboxypeptidase. See Experimental Procedures for further details.
repeating the Edman degradation of intact subunit that was reported earlier (B), we were able to reach the -Lys-Pro-Lyssequence that represented the beginning of C12. Peptide Cl4 is placed after peptide C12, since this yields alignment of the identical sequences in the two species that encompass the active-site lysyl residue at position 175 (Ref. (26) and citations therein). Fragment Cl9 must thus be located between Cl4 and C16, the latter of which was shown to be the carboxyl terminus by the absence of homoserine and by digestion with carboxypeptidase Y. The previous isolation of an active-site peptide
DISCUSSION
Based on amino acid analysis, the methionine content of ribulose-P, carboxylase from R. rubrum has been reported as 13-15 residues per 56,000-dalton subunit (7, 8). Since the NH, terminus is methionine, 13-15 cyanogen bromide fragments were anticipated. During the present study, the amino acid composition of the enzyme was checked repeatedly, and 15 + 0.3 residues of methionine were calculated. However, if the actual subunit weight of the R. rubrum enzyme is 52,700, as proven for the maize enzyme by having established its sequence (2), the calculated number of methionyl residues would become 14. Thus, the 14 cyanogen bromide fragments, either isolated or observed within other peptides, described herein appear to account for the total polypeptide chain. Consistent with this conclusion, the summation of molecular weights of these fragments is 5054.6K; this range reflects the differences in size of the four large fragments as determined by Na-dodecyl-SO,-polyacrylamide gel electrophoresis compared to amino acid analyses. We have sequenced about 70% of the total polypeptide chain (339 residues of the approximate 475). Including the tentative alignments of several small peptides (C2,
432
HARTMAN
ET
AL.
C!6 --12+
/ -,I--
--A
FIG. 9. Amino acid sequences of ribulosebisphosphase carboxylase/oxygenase from spinach (upper) and R. rubrum (lower). Numbers refer to the position of the amino acid in the spinach enzyme, based on the nucleotide sequence of the enzyme’s gene (2, 3). Alignments were made by visual inspection. Residues identical in both enzymes are enclosed in boxes. In those cases where a single base change of the spinach gene sequence could generate the amino acid found at that position in the R. rubrum enzyme, the residues are marked with horizontal bars. Gaps, attributed to deletions or insertions, appear as blank spaces. Regions of the R. rubrum enzyme not yet sequenced are denoted by a dashed line. The fragment labeled “active-site peptide” is the tryptic peptide containing the methionyl residue subject to affinity labeling by N-bromoacetyl-aminopentitol-P, (27, 28). Lys175 and Lys-334 are active-site residues based on affinity labeling with 3.bromo-1,4-dihydroxy-2butanone 1,4-bisphosphate, N-bromoacetylethanolamine phosphate, or pyridoxal phosphate (26, 2932). Abbreviations: Asp, D; Asn, N; Asx, B; Glu, E; Gln, Q; Glx, 2; Thr, T; Ser, S; Pro, P; Gly, G; Ala, A; Cys, C; Val, V; Met, M; Ile, I; Leu, L; Tyr, Y; Phe, F; His, H; Lys, K; Trp, W; Arg, R.
C3, C6a, T4B, and T4C), 94 of the 339 residues are identical to those in spinach carboxylase; an additional 102 residues could represent single base pair mutations. This degree of divergence (-72%) is in stark contrast to that between maize and spinach ribulose-Ps carboxylases in which only 49 of 475 residues are changed (2, 3). The expected usefulness of comparative sequence studies of plant and R. rubrum
carboxylases has been realized. Lysines at positions 175 and 334 in the spinach enzyme were identified as active-site residues on the basis of affinity labeling studies with 3-bromo-1,4-dihydroxy-2-butanone 1,4-bisphosphate (29), N-bromoacetylethanolamine phosphate (30), and pyridoxal phosphate (31, 32). As seen in Fig. 9, these two regions are highly conserved, and thus a catalytic role for these lysines is likely.
SEQUENCE OF RIBULOSE-P2 CARBOXYLASE
433
Lys-175 is within a 7-residue segment ysis or maintenance of structural integrity (30). which is identical in the two species, and Lys-334 is within a segment in which 9 A region of considerable interest is that residues out of 13 are identical. Recent including Lys-201, which reacts with CO, studies (26) have shown that, as with the to form a carbamate with concomitant enspinach carboxylase, Lys-175 of the corzymatic activation; this process has been responding R. rubrum enzyme is the target fully characterized in the spinach enzyme of pyridoxal phosphate. Although Lys-334 (37). Unfortunately, the corresponding lyof R. rubrum carboxylase has not been de- sine in R. rubrum carboxylase presumably rivatized by any affinity label, its assign- occurs within peptide Cl4 beyond the parment to the active site seems firm in view tial sequence elucidated and thus the comof the selective alkylation of Met-335 parison has not been made. (which is replaced by Leu in the spinach enzyme) by the affinity label 2-bromoacetylaminopentitol 1,5-bisphosphate (27, 28). REFERENCES In contrast to the illumination of func1. LORIMER, G. (1981) Annu. Reu. Plant Physiol. 32, tionally significant regions of primary 349-383. structure, these comparisons argue against 2. MCINTOSH, L., POULSEN, C., AND BOGORAD, L. an essential role of sulfhydryls in catalysis, (1980) Nature (London) 288, 556-560. for species invariance of cysteinyl residues 3. ZURAWSKI, G., PERROT, B., BOTTOMLEY, W., is not obvious. On the basis of absence of AND WHITFELD, P. R. (1981) Nucleic Acids Res. carboxymethylcysteine in peptide Cl6 and 9, 3251-3270. only one residue of carboxymethylcysteine 4. MCFADDEN, B. A., AND TABITA, F. R. (1974) BioSystems 6, 93-112. in peptides Cl2 and C14, cysteines at po5. RUTNER, A. C. (1970) Biochem. Biophys. Res. sitions 99,221,427, and 459 in the spinach Commun. 39,923-929. enzyme are not present in R. rubrum car6. SIEGEL, M. I., WISHNICK, M., AND LANE, M. D. boxylase. Cys-84, Cys-172, Cys-192, and (1972) in The Enzymes (Boyer, P. D., ed.), 3rd Cys-247 of the spinach enzyme are reed., Vol. 6, pp. 1699192, Academic Press, New placed by Tyr, Thr, Phe, and Pro, respecYork. tively, in R. rubrum carboxylase. The one 7. TABITA, F. R., AND MCFADDEN, B. A. (1974) J. remaining cysteinyl residue of spinach carBiol. Chem. 249, 3459-3464. boxylase at position 284 may correspond 8. SCHLOSS, J. V., PHARES, E. F., LONG, M. V., to the cysteinyl residue in peptide T4B NORTON, I. L., STRINGER, C. D., AND HARTMAN, F. C. (1979) J. Bacterial. 137, 490-501. from R. rubrum carboxylase (lo), but the 9. AKAZAWA, T., TAKABE, T., ASAMI, S., AND Kodegree of homology is not striking (Fig. 9). BAYASHI, H. (1978) in Photosynthetic Carbon The four cysteinyl residues at positions 38, Assimilation (Siegelman, H. W., and Hind, G., 70, 189, and 374 of R. rubrum carboxylase eds.), pp. 209-226, Plenum, New York. that can be confidently aligned with the 10. STRINGER, C. D., NORTON, I. L., AND HARTMAN, spinach enzyme are replaced by Ala, Trp, F. C. (1981) Arch. Biochem. Biophys. 208, Val, and Val, respectively. The likely ex495-501. clusion of sulfhydryl groups as partici11. LAEMMLI, U. K. (1970) Nature (London) 227, pants in the catalytic process is of special 680-685. interest in view of considerable specula- 12. SHAPIRO, A. L., VINUELA, E., AND MAIZEL, J. V. (1967) Biochem. Biophys. Res. Commun. 28, tion about such a role first stimulated by 815-820. chemical modification studies by Trown P. T., AND BURGESS, D. R. (1978) and Rabin (33) but continuing in more re- 13. MATSUDAIRA, Anal. Biochem. 87, 386-396. cent articles (34-36). Although Cys-172 R., ANDKOVACS, K. (1974) and Cys-459 in spinach carboxylase appear 14. PENKE, B., FERENCZI, Anal. Biochem. 60, 45-50. to be located in the active-site region based 15. GROSS, E. (1967) in Methods in Enzymology on their reaction with the affinity label N(Hire, C. H. W., ed.), Vol. 11, pp. 238-255, bromoacetylethanolamine phosphate (proAcademic Press, New York. vided the enzyme is deactivated), this does 16. MAHONEY, W. C., ANDHERMODSON, M. A. (1979) not dictate an essentiality in either catalBiochemistry 1S,3810m3814.
434 17. 18.
HARTMAN
MAHONEY, SON, M.
W. C., SMITH, P. K., A. (1981) Biochemistry
FONTANA, A., DALZOPPO, ZAMBONIN, M. (1981)
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20.
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T. W., MOLE, J. E., W. A., AND BENNETT,
SMITHIES, O., GOODFLIESH,
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F. C., ANDFRAIJ,
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D.,
AND
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F. C.
(1978)
80,
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SCHLOSS,
31.
F. C. (1978) J. Biol. Chem. 253, 570775711. PAECH, C., AND TOLBERT, N. E. (1978) J. Biol.
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Chem.
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C. D., ANDHARTMAN,
7864&787X
32.
SPELLMAN, M., TOLBERT, N. E., AND HARTMAN, F. C. (1979) Abstract of Papers, 178th National Meeting of the American Chemical Society,
33.
TROWN,
Washington,
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T.,
WILDNER,
36.
(1980) WILDNER,
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D. C., Sept.
P. W.,
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FONTANA,
ods in masheff,
J. F. (1978) 1135-1142.
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1048.
84,622-
10,4912-
HARTMAN,
3501-3505.
1178.
Biochem.
Res. Commun.
AND
257,
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GIBSON, D., FANNING, E. M., R. M., GILMAN, J. G., AND BAL-
E., ANDLAI,
H.-M.,
Biophys.
F. C.
102, 35-38.
J.
68, 47-53. 24.
FRAIJ,
41,
S. N., Press,
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TARR, G. E., BEECHER, MCKEAN, D. J. (1978)
LANTYNE, 4921. 23.
27.
Chem.
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I. L., AND HARTMAN, 21, 138091385.
Enzymology
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A. S., CORNELIUS, J. D., TIDWELL,
J. C. (1980) 21.
in Methods
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28.
HAYASHI,
AL.
26.
20,6997-
7004. 19.
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LORIMER, 1240.
AND
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RABIN,
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AND
BIOL
B.
R.
52,
88-93.
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SANF,,
2. Naturforsch. G. F. (1981)
3.
(1964)
P. V.,
AND
HENKEL,
35c, 239-248. Physiol. Plant. Biochemistry
J.
(Copen20,
1236-
SEQUENCE
88.9 d d
OF
RIBULOSE-P,
CARBOXYLASE
436
HARTMAN
16.1 21.4 29.0 24.1 29.0 *,.* 19.4 8.2
ET
AL.
SEQUENCE
OF
RIBULOSE-P2
437
CARBOXYLASE
8
13.Rd
9 10
8.8
8.8 5.3 4.0 2.6
e
OF BIOCHEMISTRY
219,
No.
2, December,
AND pp.
BIOPHYSICS
422-437,
1982
Purification and Sequencing of Cyanogen Bromide Ribulosebisphosphate Carboxylase/Oxygenase Rhodospirillum rubrum’ FRED Biology
Division,
C. HARTMAN,’ CLAUDE MARK I. DONNELLY,4
D. STRINGER, AND BASSAM
Fragments from
JOHN FRAIJ5
OMNAAS,”
Oak Ridge National Laboratory and the University of Tennessee-Oak School of Biomedical Sciences, Oak Ridge, Tennessee 37830 Received
June
23,
from
Ridge
Graduate
1982
As a part of the goal to determine the total sequence of Rhodospirillum rubrum ribulosebisphosphate carboxylase/oxygenase, the cyanogen bromide fragments were fractionated and sequenced (or partially sequenced). Twelve of the anticipated 14 peptides were obtained in highly purified form. The other two peptides were located, respectively, within a trytophanyl cleavage product (which overlapped with four CNBr fragments) and within an active-site peptide characterized earlier (which overlapped with three CNBr fragments). These overlaps coupled with amino and carboxyl terminal sequence information of the intact subunit and the availability of the sequence of the corresponding enzyme from higher plants permitted alignment of all fragments. Eight CNBr peptides were sequenced completely; four of the CNBr peptides consisted of more than 80 residues and were only partially sequenced as permitted by direct Edman degradation. Of the approximate 47.5 residues per subunit, 339 were placed in sequence. The lack of extensive conservation of primary structure between R. rubrum and higher plant carboxylases permits the tentative identifications of those regions likely to be functionally important.
As the enzyme that catalyzes the initial step in photosynthetic CO, fixation and in photorespiration (see Ref. (1) for review),
ribulose-PZ6 carboxylase/oxygenase (EC 7.1.1.39) plays a pivotal role in plant growth and productivity and thus has become the subject of intense structural and mechanistic studies. Because of conservation of essential molecular features during evolution, comparative sequence analyses of functionally analogous enzymes provide a useful approach to discern regions of primary structure essential either to catalysis, regulation, or integrity of
’ The U. S. Government’s right to retain a nonexclusive royalty-free license in and to the copyright covering this paper, for governmental purposes, is acknowledged. Research sponsored jointly by the Science and Education Administration of the U. S. Department of Agriculture under Grant 78-59-2472-0-1161-1 from the Competitive Research Grants Office and by the Office of Health and Environmental Research, U. S. Department of Energy, under Contract W-7405.eng-26 with the Union Carbide Corporation. * To whom all correspondence should he addressed. 3 Postdoctoral investigator supported by Suhcontract 3322 from the Biology Division of the Oak Ridge National Laboratory to the University of Tennessee. 4 Postdoctoral investigator supported by NSF Grant PCM-7908990. ’ Postdoctoral investigator supported by the U. S. 0003.9861/82/140422-16$02.00/O Copyright All rights
0 1982 by Academic Press, Inc. of reproduction in any form reserved
Department of Agriculture grant through Suhcontract, 3322 from the Biology Division of the Oak Ridge National Laboratory to the University of Tennessee. 6 Abbreviations used: Rihulose-P,, D-rihulose 1,5bisphosphate; Na-dodecyl-SO,, sodium dodecyl sulfate; DMAA, N,N-dimethyl-N-allylamine; and Quadrol, N,N,N’,N’-tetrakis(2-hydroxypropyl)-ethylenediamine. 422
SEQUENCE
OF
RIBULOSE-P,
three-dimensional structure. Although complete primary structures of the large subunits of ribulose-P, carboxylase from both maize and spinach have been deduced by cloning and sequencing their respective genes (2, 3), little is learned concerning which residues are critical to function, because these two carboxylases exhibit 90% homology. In contrast, comparisons between the carboxylases from higher plants and the purple non-sulfur bacterium Rhodospirillum rubrum appear especially appropriate as these organisms are evolutionarily quite distant (4) and their respective carboxylases differ markedly in both primary and quaternary structure. For example, ribulose-P2 carboxylase from higher plants consists of eight large (-53,000 daltons) and eight small (-14,000 daltons) subunits (2, 5, 6), whereas the enzyme from the purple nonsulfur bacterium Rhodospirillum rubrum is a simple dimer of large subunits (7, 8). Total sequence homology appears to be no greater than 20% (9, 10); thus, stretches of homology that are found are likely to be crucial to function. We previously determined the sequence of cysteine-containing peptides from tryptic digests of R. rubrum ribulose-P2 carboxylase in order to ascertain if any cysteinyl residues were species invariant; because homologies were not observed, we suggested that sulfhydryl groups are not essential to catalysis (10). We now report purification and sequencing of cyanogen bromide fragments, which account for virtually the entire polypeptide chain; about 70% of the total sequence has been obtained. Two lysyl residues previously implicated at the active site are observed to be located in highly conserved regions. EXPERIMENTAL
PROCEDURES
Materials. Ribulose-P2 carboxylase/oxygenase from R. rubrum was purified to homogeneity according to a published procedure (8). Cyanogen bromide and polybrene were obtained from Aldrich Chemical Company. Other chemicals and vendors were iodosobenzoic acid from Pierce, Carboxypeptidase Y from Sigma, ultrapure urea from Schwarz/Mann, pepstatin from United States Biochemical, and [3H]iodoacetic acid from New England Nuclear. Chemicals, buffers, and reagents required for amino acid analyses and for
CARBOXYLASE
423
automated Edman degradations were purchased from either Pierce Chemical Company or Beckman Instruments, Inc. Ingredients for Na-dodecyl-SO,-polyacrylamide gel electrophoresis were products of Bethesda Research Laboratories. General methods. Preceding cleavage with either cyanogen bromide or iodosobenzoic acid, the carboxylase was carboxymethylated with [3H]iodoacetic acid (sp act = 1.7. lo6 cpm/wmol) as described earlier (10). The alkylated protein had a sp act of 9.5.10s cpm/ pmol representing an incorporation of 5.6 mol of carboxymethyl groups per mole of subunit. Urea was deionized by batchwise treatment with Bio-Rad AC, 501-X8. A Packard 3255 liquid scintillation spectrometer was used to measure radioactivity. Samples were diluted to 0.5 ml with water and then dissolved in 10 ml of ACS counting solution (Amersham Corporation). During column chromatography, peptides were detected by continuous monitoring of effluents’ absorbancies at 206,215, or 280 nm with a LKB Uvicord III or a Gilson Holochrome. Peptides were dialyzed in Spectrapor 2000 membrane tubing (Spectrum Medical Industries). Electrophoresis. Na-dodecyl-SO,-polyacrylamide slab gel electrophoresis was carried out according to procedures of either Laemmli (11) or Shapiro et al. (12). For the monitoring of peptides during their purification, the Laemmli (11) system was used with a microslab apparatus (13) available commercially from Idea Scientific, Corvallis, Oregon. The acrylamide concentration was 15%, and the gel dimensions were 100 mm in width X 90 mm in height X 0.5 mm in thickness. A slab gel apparatus from Bethesda Research Laboratories (Model V16) was used according to the manufacturer’s instructions [slight modifications of those of Shapiro et al. (12)] to estimate molecular weights of peptides. The acrylamide concentration was 15%, and the gel dimensions were 16.8 cm in width X 16 cm in height X 3 mm in thickness, In all cases, peptides were visualized with Coomassie blue (0.1%) in methanol:water:acetic acid (5:5:1); the same solvent was used for destaining. Amino acid analyses. Total acid hydrolysis of peptides were achieved in evacuated (-50 pm Hg) sealed tubes with 6 N HCl/O.Ol M 2-mercaptoethanol at 110°C for 21 h. Hydrolysates were dried on a Speed Vat Concentrator (Savant Instruments Inc.) and subjected to chromatography on a Beckman 121M amino acid analyzer using Beckman’s “3-hour-single-column system.” The analyzer was hard wired to a PDP data acquisition system. Computer programs used in data collection and analysis were provided by S. S. Stevens and J. T. Holderman of the Oak Ridge National Laboratory. Separate samples were hydrolyzed with 3 N mercaptoethanesulfonic acid for the determination of tryptophan content (14); hydrolysates were brought to pH 2.0 with 3 N NaOH and diluted lo-fold with H,O prior to analysis.
424
HARTMAN
Digestion with cyanogen bromide (15). To a solution of carboxymethylated ribulose-P, carboxylase (430 mg, 7.7 pmol of subunit) in 40 ml of 70% (v/v) formic acid was added 400 mg (3.8 mmol) of CNBr dissolved in 2 ml of 70% formic acid. The digestion was allowed to proceed in the dark at room temperature for 20 h at which time the reaction was terminated by the addition of H,O (600 ml) and lyophilization of the diluted solution to dryness. The sample was redissolved in 60 ml of 0.1 N acetic acid, subdivided into aliquots containing 40 mg of digested protein, lyophilized, and then stored at -80°C. Digestion with iodosobenzoic acid (16-18). Iodosobenzoic acid (170 mg) was added to a solution of carboxymethylated ribulose-Py carboxylase (86 mg, 1.54 pmol) consisting of 80% (v/v) acetic acid (10 ml), 8 M guanidine hydrochloride (1.8 ml), and thiodiglycol (45 ~1). After 5 min in the dark at room temperature, the reaction mixture was quenched by the addition of 2-mercaptoethanol (660 ~1) and desalted on a 2.5 X 19-cm column of Bio-Gel P-2. The excluded peptide region was dried by lyophilization. Digestion with carboxypeptidase Y (19). Commercial enzyme (10 mg) was partially purified by gel filtration at 4°C on a 2 X 82.cm column of Bio-Gel P-100 equilibrated with 0.01 M Na2HP0,/0.1 M NaCl/l mM EDTA (pH 7.0) (19). To the peak fraction (2 ml, 14 @M in carboxypeptidase) was added 20 ~1 of pepstatin (50 mg/ml in dimethyl sulfoxide). Digestion of peptides with carboxypeptidase Y was carried out at room temperature in 0.1 M pyridine acetate (pH 5.5) containing norleucine as internal standard; other specific conditions are provided in figure legends. Periodically, loo-p1 aliquots of the digestion mixtures were diluted into 2 ml of boiling water; the diluted aliquots were centrifuged to remove insoluble material, and the supernatants were lyophilized to dryness, dissolved in 300 ~1 of 0.1 M sodium citrate (pH 2.2), and then subjected to amino acid analyses. All calculations were normalized to the norleucine internal standard. Sequence analyses. Purified peptides (36-190 nmol) were subjected to automated Edman degradations with a Beckman 890C sequencer, the vacuum system of which was modified according to Bhown et al. (20). A liquid nitrogen cold trap was inserted between the low vacuum pump and the vacuum manifold. Polybrene (2 mg) was added to the peptide to reduce its extraction from the reaction cup (21). Both dimethylallylamine and Quadrol buffer systems were employed; Beckman’s peptide program 102974 was used with the former. The program used with the Quadrol buffer was that of Bhown et al. (20) with several modifications. The buffer concentration was reduced from 0.5 to 0.1 M, the drying step after delivery of phenyl isothiocyanate was increased from 20 to 60 s, and the coupling steps and drying steps after delivery of benzene/ethyl acetate were from Beckman’s peptide program 030176. Generally, half of the fraction from each
ET
AL.
cycle was hydrolyzed in base for quantitation as free amino acid on the amino acid analyzer (22). Threonine and arginine appear as ol-aminobutyric acid and ornithine, respectively, in base hydrolysates. The other half of each fraction was converted to the phenylthiohydantoin for identification by high-performance liquid chromatography (Laboratory Data Control); this provided confirmation of the identifications made by amino acid analyses and distinguished aspartic acid and glutamic acid from the corresponding amides. Tentative identifications of PTH-serine based on HPLC and the presence of a degradation product in the base hydrolysates that coelutes with cysteic acid on the amino acid analyzer were confirmed by carrying out acid hydrolysis on a portion of the PTH derivative thereby converting serine to alanine (23). For those peptides that were radioactive, 10% of each fraction from the sequencer was counted so as to identify carboxymethylcysteine; the remainder of the fraction was then processed as already described. RESULTS
Purification of CNBr fragments. Initial fractionation of CNBr fragments was achieved by ion-exchange chromatography on carboxymethyl cellulose at pH 4.0 in the presence of 8 M urea (Fig. 1). Each peak was inspected by Na-dodecyl-SO,polyacrylamide gel electrophoresis; all of the components that eluted before initiation of the salt gradient were small peptides as they could not be visualized by staining the gels with Coomassie blue. Peaks C8, C9, ClO, Cll, C17, and Cl8 represented complex mixtures of fragments larger than 14,000 daltons, which were assumed to be incomplete digestion products and were thus not processed further. The four major large fragments in the CNBr digest (see Fig. 2, lanes 2 and 7) were found in the regions denoted C12-C13, C14-C15, C16, and C19. Fractions from these regions were separately pooled, and the four pools were dialyzed against 0.5% (v/v) acetic acid and then lyophilized to dryness. The lyophilized samples were dissolved in 8 M urea buffered at pH 5.0 with 0.01 M ammonium acetate and rechromatographed on carboxymethyl cellulose (Fig. 3). The center fractions of the four major peaks were pooled as indicated in the figure, dialyzed against 0.5% acetic acid, and lyophilized. Each of the four large peptides (denoted C12, C14, C16, and C19) thus
SEQUENCE
OF
RIBULOSE-Ps
425
CARBOXYLASE
0 0
b
Ii0
150*0
2fil4oto
io
do
VOLUME (ml)
FIG. 1. Ion-exchange chromatography of a cyanogen bromide digest of “H-carboxymethylated ribulose-P, carboxylase on Whatman CM-52 carboxymethyl cellulose. The sample (46 mg) in 2.0 ml of 0.01 M ammonium formate/ M urea (pH 4.0) was applied to the column (1 X 50 cm) equilibrated with the same ammonium formate/urea buffer. The column was eluted first with 150 ml of equilibration buffer followed by two successive 400.ml linear gradients (O-0.1 M NaCl and 0.1-0.2 M NaCl) prepared with the same urea-containing equilibration buffer.
obtained appeared highly pure by gel electrophoresis (Fig. 2). Based on mobilities relative to those of the standards also
12345678
FIG. 2. Na-dodecyl-SO,-polyacrylamide gel electrophoresis of the total cyanogen bromide digest of carboxymethylated ribulose-P, carboxylase (lanes 2 and 7) and the purified large fragments Cl2 (lane 3), Cl9 (lane 4), Cl4 (lane 5), and Cl6 (lane 6). A standard from Pierce containing soybean trypsin inhibitor (21.5K). cytochrome c (12.5K), aprotinin (6.5K), and insulin B chain (3.4K) is shown in lanes 1 and 8. See Experimental Procedures for further details.
shown in Fig. 2, peptides C12, C14, C16, and Cl9 have molecular weights of ll.lK, 8.2K, 7.8K, and 9.4K, respectively (Fig. 4). The smaller peptides represented by peaks C2 through C7 (Fig. 1) were all purified by essentially the same proceduresfirst, gel filtration on Sephadex G-25, and second, when necessary, ion-exchange chromatography on DEAE-cellulose. In the case of C3, gel filtration on a G-25 column in the presence of 6 M urea was necessary to remove a contaminating peptide whose amino acid composition was the same as C5; C3 was then freed of urea by desalting on a G-25 column equilibrated with 0.01 M NH,HC03 (pH 8.0). Lyophilization served to concentrate and desalt all peptides obtained from DEAE and G25 columns when NH,HCO, (in the absence of urea) was the buffer. Peptide material could not be detected in peak Cl (Fig. 1) by amino acid analysis suggesting that this peak represents a change in refractive index associated with the solvent front. Purifications of peptides C2, C3, C4a, C4b, C5, C6a, C6b, and C7 are shown in Figs. 5 and 6. Gel filtration of peaks C2 and C3 combined yielded C2 as a pure peptide (Fig. 5A). Peptide C3 was obtained from the pool of C3 plus C5 (Fig. 5A) by gel filtration in the presence of urea which removed peptide C5 (Fig. 5B). Amino acid analyses of the initial two peaks in Fig. 5A
426
HARTMAN
0.2
64
ET
AL.
a4
0.07
_.^
Cl4
0.07
0.6-
I 0.20
CG
i ;
1
/’ 27
7
0.16
/ /’ 169
243
400
27
61
135
169
243
400
FIG. 3. Rechromatography of the four large cyanogen bromide fragments (C12, C14, C16, and C19) on a column (1 X 50 cm) of Whatman CM-52 carboxymethyl cellulose equilibrated with 0.01 M ammonium acetate/8 M urea (pH 5.0). In all four cases the column was eluted with a 400-ml linear gradient of sodium chloride dissolved in the urea-containing equilibration buffer. The final sodium chloride concentration was as indicated in each panel.
suggested that these corresponded to peptides C6b and C4a in impure forms. Chromatography of peptide C4 (obtained from gel filtration, Fig. 4C) on DEAE-cellulose resolved peptides C4a and C4b (Fig. 6A). Peak C5 chromatographed as one major component on both G-25 and DEAE-cellulose (Fig. 5D and Fig. 6B). Peak C6 was resolved into peptides C6a and C6b by gel filtration (Fig. 5E) and each of these was purified further on DEAE-cellulose (Figs. 6C and D). A single peptide was obtained from peak C7 (Figs. 5F and 6E). Purification of two trytophanyl cleavage products. The lyophilized digest of the car-
boxylase (86 mg) that had been treated with o-iodosobenzoic acid (see Experimental Procedures) was extracted with 10 ml of 10 mM acetic acid, and the insoluble material was removed by low-speed centrifugation. The supernatant was brought
to 60 mM ammonium bicarbonate thereby resulting in the precipitation of additional
20.~ a
,o-1 ‘& F * 5-
a\ 4
31 4
5
6 7 MIGRATION
e 9 DISTANCE (cm)
IO
4
I1
FIG. 4. Determination of molecular weights of the large cyanogen bromide fragments based on the gel shown in Fig. 2. The open circles represent the standard proteins: soybean trypsin inhibitor (21.5K), cytochrome c (12.5K), aprotinin (6.5K), and insulin B chain (3.4K).
SEQUENCE
OF
RIBULOSE-P2
,6 , 1
I-: >
/60 I I 30 T s d 2 b x
427
CARBOXYLASE
the sample was subjected to gel filtration on Sephadex G-50 (Fig. 7B). The second peak (12) was purified by cation-exchange chromatography on SP-Sephadex (Fig. 7C). Amino acid compositions of purified peptides. Results from amino acid analyses of the purified peptides are provided in Table I along with the percentage recoveries of each peptide, estimates of total number of residues and molecular weights, and specific radioactivities of those five peptides containing carboxymethyl cysteine. The isolation of five labeled CNBr peptides with similar specific activities, about onefifth that of the intact polypeptide (see Experimental Procedures), is consistent with the presence of only one residue of carboxymethyl cysteine in each and ac-
E
i
‘2
A
A
c4a C4b
20 IO
20 10
140
180 VOLUME
220
i
!6(1
0.4 Am
(ml1
FIG. 5. Gel filtration of peaks C2 + C3 (A and B), C4 (C), C5 (D), C6 (E), and C7 (F) that were obtained from the CM-cellulose column (see Fig. 1). The column (1.7 x 220 cm) consisted of Sephadex G-25 equilibrated and eluted with 0.01 M NH,HCOB (pH 8.0). For the resolution of peptides C5 and C3 (B) the NH,HCO? buffer contained 6 M urea.
0.2 P
8 ‘, 5
/’ .0’ E
peptides which were again removed by centrifugation. The supernatant, which contained peptides soluble in 60 mM ammonium bicarbonate, was fractionated on Sephadex G-25 (Fig. 7A). Rechromatography of peptide 11 on the same column yielded this material in a highly pure form. Fractions from the excluded region (Fig. 7A) were lyophilized; the residue was dissolved in 0.5 ml of 8 M guanidine hydrochloride, and following the addition of HZ0 (1 ml)
c7 :\
04 0.2
,’
,’ i c, _I,.-
,’
,’
,’
0.4
- ’ ’
0.2
m 100
200 VOLUME
300
400
irnl)
FIN;. 6. Ion-exchange chromatography of peptides after their subjection to gel filtration (see Fig. 5). The column (1 X 25 cm) consisted of Whatman DE-52 DEAE-cellulose equilibrated with 0.01 M NH,HCOB (pH 8.0) and eluted with 400.ml linear gradients of NH,HCO, (pH 8.0) as specified by the ordinates.
428
HARTMAN
counts for the five cysteinyl residues per subunit (10). In the smaller peptides, the absence of certain amino acids and the approximate integral ratios of residues indicate a high degree of purity. Amino acid analyses of the four large fragments (Cl2, C14, C16, and C19) are not informative concerning degree of purity; however, as already indicated, these peptides were homogenous asjudged by acrylamide gel electrophoresis (Fig. 2). The molecular weights of peptides C14, C16, and Cl9 calculated from amino acid analyses were somewhat greater than those determined by gel electrophoresis. The amino acid composition of peptide C7 was identical to that of peptide C6b except the former contained one residue of methionine. Carboxyl terminal analyses of intact subunit, peptide C16, and peptide Il. Because of the absence of homoserine in peptide C16, it was assumed to be the carboxyl terminal fragment. Analyses of this fragment and the intact carboxymethylated subunit by digestion with carboxypeptidase Y were consistent with a carboxyl terminal sequence of Ala-Leu-Pro-Ala (Figs. 8A, B). Although leucine and proline were released at very similar rates, proline is placed as the penultimate residue on the basis of alanine, in low yield, as the only product of digestion with carboxypeptidase A (data not shown). The excess leucine observed during digestion of carboxymethylated subunit with carboxypeptidase Y is likely a consequence of a leucine-specific endopeptidase which contaminates commercial preparations (19, 24). Greater than stoichiometric amounts of leucine and many other amino acids were released when peptide Cl6 was digested with carboxypeptidase Y that had not been subjected to gel filtration followed by treatment with the endopeptidase inhibitor pepstatin as described under Experimental Procedures. Furthermore, without these precautions extensive degradation of Cl6 into multiple discrete peptides was observed by Na-dodecyl-Sod-gel electrophoresis. Since peptide 11, obtained by fragmentation of the whole subunit with iodosobenzoate, lacked the characteristic absorbance of oxidized indole (25),
ET AL.
FIG. 7. Purification of two tryptophanyl cleavage peptides from ribulose-Pp carboxylase. (A) The mixture of peptides that were soluble in ammonium bicarbonate (see Results) was fractionated on a 1.5 X 230.cm column of Sephadex G-25 equilibrated with 0.01 M NH,HCOs (pH 8.0). Peptide II was rechromatographed on the same column (profile not shown). (B) Peptide I2 from (A) was subjected to gel filtration on a 1.5 X 215cm column of Sephadex G-50 equilibrated with 0.01 M NH,HCOs (pH 8.0). (C) Peptide I2 from (B), after lyophilization, was redissolved in 2.0 ml of 6 M urea/32 mM H,PO, (pH 3.0) and subjected to cation-exchange chromatography on a 1 X 20.cm column of Sulfopropyl Sephadex C-25. The column was equilibrated with 6 M urea/32 mM H,PO, (pH 3.0) and eluted with a 400.ml linear sodium chloride gradient (O-1.3 M) prepared with the same ureacontaining buffer.
it too was tentatively identified as a carboxyl-terminal segment. Digestion with carboxypeptidase Y confirmed the -LeuPro-Ala-COOH sequence (Fig. 8C). Sequence analyses of purified peptides. Automatic Edman degradations were carried out on the purified peptides (Tables I-XIV in miniprint supplement); the lack of a second (or multiple) residue at cycle one in quantities greater than 5% of the residue identified as the amino terminus verified the high degree of purity of these peptides. Although the results for only a single analysis of each peptide are shown, the established sequences (or partial se-
2.2 (2)
0.1
GUY Ala
MI
Recovery
Calcd.
residues
Total
A%
TOP
LYS
His
TY~ Phe
28
24
50
11
1077
9
996
6
0.2
4.3 (2)
-
2.3 (1)
0.2
2.3 (1)
2.1 (1)
0.1
623
1.1 (1)
2.1 (2)
1.2 (1)
Ile
Leu
-
Met
(%)
1.1 (1)
1.2 (1)
Glu Pro
0.1
2.0 (1)
0.1
0.1
Val
3.9 (2)
2.0 (2)
0.1
Ser
1.0 (1) -
0.1
1.6 (1)
2.2 (1)
2.1 (2)
0.8 (1)
C4a
1.0 (1)
c3
Cys (Cm) Asp Thr
c2
Homoserine
Amino acid
20
1328
12
1.6 (1)
1.4 (1)
8 912 56
18 34
_
1.1 (1)
1.0 (1)
2074
1.4 (1)
1.6 (1)
1.4 (1)
1.4 (1)
1.6 (1)
0.1
20
3240
29
1.1 (1)
1.2 (1)
1.0 (1)
2.0 (2)
4.2 (4)
2.1 (2)
0.2
2.1 (2)
3.2 (3)
1.0 (1)
1.4 (1) 1.0 (1)
2.3 (2)
4.6 (4)
2.1 (2)
3.3 (3)
1.1 (1)
0.8 (1)
C6b
1.0 (1)
1.0 (1)
0.9 (1)
0.9 (1)
C6a
3.8 (3)
1.4 (1)
5.0 (4)
1.3 (1)
1.3 (1)
1.4 (1)
1.2 (1)
C5
9
3371
30
1.1 (1)
1.3 (1)
1.0 (1)
-
2.0 (2)
4.2 (4)
2.1 (2)
0.8 (1)
2.1 (2)
3.2 (3)
2.3 (2)
4.6 (4)
2.1 (2)
6.0 (6)
4.7 (5)
14 25
1401
29
11.8K
110
4.6 (5)
6.2 (6)
1.9 (1)
1.9 (2)
1.8 (1)
0.1
2.0 (2)
6.0 (6)
-
8.4 (8) 15.1 (15)
12.4 (12) 13.4 (13)
35
lO.OK
92
51
9.5K
88
29
10.8K
98
8.0 (8)
1.8 (2)
6.8 (7)
3.2 (3)
2.2 (2) 2.7 (3)
5.2 (5) 4.0 (4)
5.0 (5)
6.0 (6)
5.0 (5)
5.3 (5) 4.0 (4)
7.0 (7) 2.0 (2)
3.6 (4)
2.8 (3)
7.8 (7)
3.1 (3)
6.4 (6)
4.0 (4)
8.6 (9)
4.0 (4)
6.9 (7)
8.0 (8)
1.1 (1)
0.9 (1)
Cl9
8.4 (8)
3.4 (3)
2.0 (2)
9.2 (9)
Cl6
2.0 (2) 1.8 (2)
9.2 (9)
5.2 (5)
2.0 (1)
4.4 (4)
7.0 (7)
10.0 (10)
3.3 (3)
11.3 (11)
17.0 (17)
9.4 (9)
8.3 (8)
3.4 (4) 9.4 (9)
7.2 (7)
2.8 (3)
2.0 (2)
10.4 (IO)
0.9 (1)
0.7 (1)
Cl4
8.6 (9)
3.1 (3)
11.3 (11)
12.4 (12)
1.3 (1)
0.9 (1)
Cl2
2.0 (1)
-
~
3.9 (2)
5.8 (3)
2.0 (1)
1.9 (1)
3.8 (2)
0.1
3.3 (3) -
-
1.8 (1)
Cl3
1.1 (1)
1.0 (1)
C7
Peptide (nmol found and number of residues in parentheses)
0.1
1.4 (1)
0.02
3.1 (2)
1.6 (1)
2.9 (2) -
2.9 (2)
1.5 (1)
1.4 (1)
C4b
I
AMINO ACID COMPOSITIONSOF PURIFIED FRAGMENTS
TABLE
32
1584
15
2.5 (2)
1.3 (1)
-
~
2.5 (2)
-
1.4 (1)
3.8 (3)
1.5 (1)
1.3 (1)
1.4 (1)
1.4 (1)
1.3 (1)
1.4 (1)
-
I1
8
5449
56
1.2 (2)
0.6 (1)
0.6 (1)
1.7 (3)
2.2 (4)
2.3 (4)
1.6 (3)
1.0 (2)
4.6 (8)
8.1 (13)
1.9 (3)
1.1 (2)
1.0 (2)
1.3 (2)
3.2 (5)
0.4 (1)
I2
HARTMAN
430
ET
TABLE SEQUENCE
AL.
II
OF PURIFIED
FRAGMENTS’
Peptide c2
Gly-Asp-Asp-Glu-G;y-Hse
c3
Ile-Ala-Ser-Phe-Leu-Thr-Leu-Thr-Hse
C4a
Lys-Ala-Cys-Thr-P:o-Ile-Ile-Ser-Gly-G!y-Hse
C4b
Glu-Gly-Glu-Ser-&-Asp-Arg-AlaIle-;!a-Tyr-Hse
c5
Leu~Thr~Gln-Asp-G;u-Ala-Glu-Gly-Pro-~~e-Ty~-A~g-Gl”-Se~-~~-Gly-Gly-Hse
C6a
Gly-Asp-Val-Glu-Tir-
C6b
Asp-Gln-Ser-Ser-A~g-Tyr-Val-Asn-Le~-~~a-Le~-Lys-Gl~-Gl~-~~p-Le~-Ile-Ala-Gly-~~y25 Glu-His-Val-Leu-Cys-Ala-Tyr-Ile-Hse
Cl3
Ala-Arg-Leu-Gln-G~y-Ala-Ser-Gly-Ile-~~s-Th~-Gly-Th~-Hse
Cl2
Lys-Pro-Lys-Ala-G~y-TyrGly-Tyr-Val-~~a-Th~-Ala-Ala-His~~~e-Ala-Ala-Gl~-Se~-~~~25 3” Thr~Gly~Thr-Asp-Val-Glu-Val-Cys-Thr-Thr-Asp-Asp-Phe-Th~~~~g-Gly-Val-Asx-Ala-L~~ 1.5 -Val-Tyr-Glx-Val-Asx-Gly-Ala-----
Cl4
His-Asp-Phe-Tyr-V~l-Pro-Glu-Ala-Ty~-~~g-Ala-Le”-Phe-Asp-~~y-P~o-Se*-Val-Asn-~25 30 Ser-Ala-Leu-Trp-Lys-Val-Leu-Gly-Arg-Pro-Gl~-Val-Asp-Gly-Gly-Le~-Val-Val-Gly-~~~45 Ile-Ile-Lys-Pro-Lys-Leu-Gly-Leu-Arg-~~o-----
Cl6
Pro-Gly-Phe-Phe-G~n-Asn-Leu-GlyAsn-~~a-Asn-Val-Ile-Le~-~~~-Ala-Gly-Gly-Gly-~~a-
Ala-Lys-Hse
35
Phe-Gly-His-Ile-~~x-Gly-Pro-Val-Ala-~~y-Ala-A~g-Se~-Le~-~y-Glx-Ala-T~-Glx------~ (COOH-terminus Cl9
fragment)
Arg-Arg-Ala-Gln-A~p-Glu-Thr-Gly-Glu-~~a-Lys-Leu-Phe-Ser-A$-Asn-Ile-Thr-Ala~~~pAsp-Pro-Phe-Glu-~-Ile-Ala-Arg-Gly-~~x-Tyr-Val-Leu-Le~-~~~-Phe-Phe-Gly-AsxA”$--------
11
Arg-Lys-Ala-Leu-C;;y-Val-Glu-Asp-Th~-~~g-Se*-Ala-Leu-Pro-A~a
12
Gly-Gly-Met-Lys-A~a-Cys-Thr-Pro-Ile-~~-Se~-Gly-Gly-Met-d~n-Ala-Leu-Arg-Met-~~o-
/
Gly-Phe-Phe-Gln-A~n-Leu-Gly-Asn-Ala-A~n-Val----’ Cysteinyl
residues
are carboxymethylated;
Hse,
homoserine.
quences) (Table II) were confirmed by duplicate degradations. Peptide C7 differed from peptide C6b only in having a methionine added to the amino terminus (data not shown). This sequence is identical to that determined earlier by Edman degradation of the intact subunit (8); thus, C6b and C7 are both derived from the amino end of the subunit and the latter is a consequence of incomplete cleavage of the amino terminal methionyl residue. Alignment of fragments. A comparison of the partial sequence of the R. rubrum
carboxylase and the total sequence of the spinach enzyme large subunit, which was determined earlier (3), is illustrated in Fig. 9. The alignments of the four large CNBr fragments are straightforward. The amino terminal fragment C6b is contiguous with fragment Cl2 based on obvious homology from positions 53 to 72;7 furthermore, by 7 Because of deletions and insertions, the two carboxylases are not exactly the same length; however, for simplicity the residue numbers of the spinach carboxylase are applied to the R. rubrum carboxylase.
SEQUENCE
OF
RIBULOSE-Ps
431
CARBOXYLASE
from the R. rubrum enzyme containing Met-335 (27, 28), which is selectively alkylated with an affinity label, provides an overlap for C13, for a fragment with the sequence Gly-Phe-Gly-Lys-Met (which was not isolated from the CNBr digest), and for C4b. Fragment 12, obtained by digestion of the enzyme with iodosobenzoic acid, overlaps four peptides: C5, C4a, a fragment with the sequence Asn-AlaLeu-Arg-Met (which was not isolated from the CNBr digest), and C16. The peptides in Fig. 9 designated with the letter “T” represent carboxymethyl cysteinylcontaining peptides from tryptic digests characterized previously (10). Placements of peptides C2, C3, C6a, T4B, and T4C are based on apparent homologies and/or seemingly proper lengths and therefore must be considered as tentative. r
10
xl TIME (mm)
30
FIG. 8. Digestion of peptide Cl6 (A), intact carboxymethylated ribulose-P, carboxylase (B), and peptide I1 (C) with carboxypeptidase Y. Digestions were carried out at room temperature in 0.1 M pyridine acetate (pH 5.5); pepstatin, dimethyl sulfoxide, and norleucine were also present at final concentrations of 500, 800, and 200 pM, respectively. In the cases of peptide Cl6 and intact subunit, the pyridine acetate buffer contained 5 M urea as a solubilizer. Final concentrations of peptides and carboxypeptidase Y in the digestion mixtures were as follows: 89 FM Cl6 with 0.64 pM carboxypeptidase, 82 FM whole subunit with 0.55 WM carboxypeptidase, and 79 pM I1 with 4.4 pM carboxypeptidase. See Experimental Procedures for further details.
repeating the Edman degradation of intact subunit that was reported earlier (B), we were able to reach the -Lys-Pro-Lyssequence that represented the beginning of C12. Peptide Cl4 is placed after peptide C12, since this yields alignment of the identical sequences in the two species that encompass the active-site lysyl residue at position 175 (Ref. (26) and citations therein). Fragment Cl9 must thus be located between Cl4 and C16, the latter of which was shown to be the carboxyl terminus by the absence of homoserine and by digestion with carboxypeptidase Y. The previous isolation of an active-site peptide
DISCUSSION
Based on amino acid analysis, the methionine content of ribulose-P, carboxylase from R. rubrum has been reported as 13-15 residues per 56,000-dalton subunit (7, 8). Since the NH, terminus is methionine, 13-15 cyanogen bromide fragments were anticipated. During the present study, the amino acid composition of the enzyme was checked repeatedly, and 15 + 0.3 residues of methionine were calculated. However, if the actual subunit weight of the R. rubrum enzyme is 52,700, as proven for the maize enzyme by having established its sequence (2), the calculated number of methionyl residues would become 14. Thus, the 14 cyanogen bromide fragments, either isolated or observed within other peptides, described herein appear to account for the total polypeptide chain. Consistent with this conclusion, the summation of molecular weights of these fragments is 5054.6K; this range reflects the differences in size of the four large fragments as determined by Na-dodecyl-SO,-polyacrylamide gel electrophoresis compared to amino acid analyses. We have sequenced about 70% of the total polypeptide chain (339 residues of the approximate 475). Including the tentative alignments of several small peptides (C2,
432
HARTMAN
ET
AL.
C!6 --12+
/ -,I--
--A
FIG. 9. Amino acid sequences of ribulosebisphosphase carboxylase/oxygenase from spinach (upper) and R. rubrum (lower). Numbers refer to the position of the amino acid in the spinach enzyme, based on the nucleotide sequence of the enzyme’s gene (2, 3). Alignments were made by visual inspection. Residues identical in both enzymes are enclosed in boxes. In those cases where a single base change of the spinach gene sequence could generate the amino acid found at that position in the R. rubrum enzyme, the residues are marked with horizontal bars. Gaps, attributed to deletions or insertions, appear as blank spaces. Regions of the R. rubrum enzyme not yet sequenced are denoted by a dashed line. The fragment labeled “active-site peptide” is the tryptic peptide containing the methionyl residue subject to affinity labeling by N-bromoacetyl-aminopentitol-P, (27, 28). Lys175 and Lys-334 are active-site residues based on affinity labeling with 3.bromo-1,4-dihydroxy-2butanone 1,4-bisphosphate, N-bromoacetylethanolamine phosphate, or pyridoxal phosphate (26, 2932). Abbreviations: Asp, D; Asn, N; Asx, B; Glu, E; Gln, Q; Glx, 2; Thr, T; Ser, S; Pro, P; Gly, G; Ala, A; Cys, C; Val, V; Met, M; Ile, I; Leu, L; Tyr, Y; Phe, F; His, H; Lys, K; Trp, W; Arg, R.
C3, C6a, T4B, and T4C), 94 of the 339 residues are identical to those in spinach carboxylase; an additional 102 residues could represent single base pair mutations. This degree of divergence (-72%) is in stark contrast to that between maize and spinach ribulose-Ps carboxylases in which only 49 of 475 residues are changed (2, 3). The expected usefulness of comparative sequence studies of plant and R. rubrum
carboxylases has been realized. Lysines at positions 175 and 334 in the spinach enzyme were identified as active-site residues on the basis of affinity labeling studies with 3-bromo-1,4-dihydroxy-2-butanone 1,4-bisphosphate (29), N-bromoacetylethanolamine phosphate (30), and pyridoxal phosphate (31, 32). As seen in Fig. 9, these two regions are highly conserved, and thus a catalytic role for these lysines is likely.
SEQUENCE OF RIBULOSE-P2 CARBOXYLASE
433
Lys-175 is within a 7-residue segment ysis or maintenance of structural integrity (30). which is identical in the two species, and Lys-334 is within a segment in which 9 A region of considerable interest is that residues out of 13 are identical. Recent including Lys-201, which reacts with CO, studies (26) have shown that, as with the to form a carbamate with concomitant enspinach carboxylase, Lys-175 of the corzymatic activation; this process has been responding R. rubrum enzyme is the target fully characterized in the spinach enzyme of pyridoxal phosphate. Although Lys-334 (37). Unfortunately, the corresponding lyof R. rubrum carboxylase has not been de- sine in R. rubrum carboxylase presumably rivatized by any affinity label, its assign- occurs within peptide Cl4 beyond the parment to the active site seems firm in view tial sequence elucidated and thus the comof the selective alkylation of Met-335 parison has not been made. (which is replaced by Leu in the spinach enzyme) by the affinity label 2-bromoacetylaminopentitol 1,5-bisphosphate (27, 28). REFERENCES In contrast to the illumination of func1. LORIMER, G. (1981) Annu. Reu. Plant Physiol. 32, tionally significant regions of primary 349-383. structure, these comparisons argue against 2. MCINTOSH, L., POULSEN, C., AND BOGORAD, L. an essential role of sulfhydryls in catalysis, (1980) Nature (London) 288, 556-560. for species invariance of cysteinyl residues 3. ZURAWSKI, G., PERROT, B., BOTTOMLEY, W., is not obvious. On the basis of absence of AND WHITFELD, P. R. (1981) Nucleic Acids Res. carboxymethylcysteine in peptide Cl6 and 9, 3251-3270. only one residue of carboxymethylcysteine 4. MCFADDEN, B. A., AND TABITA, F. R. (1974) BioSystems 6, 93-112. in peptides Cl2 and C14, cysteines at po5. RUTNER, A. C. (1970) Biochem. Biophys. Res. sitions 99,221,427, and 459 in the spinach Commun. 39,923-929. enzyme are not present in R. rubrum car6. SIEGEL, M. I., WISHNICK, M., AND LANE, M. D. boxylase. Cys-84, Cys-172, Cys-192, and (1972) in The Enzymes (Boyer, P. D., ed.), 3rd Cys-247 of the spinach enzyme are reed., Vol. 6, pp. 1699192, Academic Press, New placed by Tyr, Thr, Phe, and Pro, respecYork. tively, in R. rubrum carboxylase. The one 7. TABITA, F. R., AND MCFADDEN, B. A. (1974) J. remaining cysteinyl residue of spinach carBiol. Chem. 249, 3459-3464. boxylase at position 284 may correspond 8. SCHLOSS, J. V., PHARES, E. F., LONG, M. V., to the cysteinyl residue in peptide T4B NORTON, I. L., STRINGER, C. D., AND HARTMAN, F. C. (1979) J. Bacterial. 137, 490-501. from R. rubrum carboxylase (lo), but the 9. AKAZAWA, T., TAKABE, T., ASAMI, S., AND Kodegree of homology is not striking (Fig. 9). BAYASHI, H. (1978) in Photosynthetic Carbon The four cysteinyl residues at positions 38, Assimilation (Siegelman, H. W., and Hind, G., 70, 189, and 374 of R. rubrum carboxylase eds.), pp. 209-226, Plenum, New York. that can be confidently aligned with the 10. STRINGER, C. D., NORTON, I. L., AND HARTMAN, spinach enzyme are replaced by Ala, Trp, F. C. (1981) Arch. Biochem. Biophys. 208, Val, and Val, respectively. The likely ex495-501. clusion of sulfhydryl groups as partici11. LAEMMLI, U. K. (1970) Nature (London) 227, pants in the catalytic process is of special 680-685. interest in view of considerable specula- 12. SHAPIRO, A. L., VINUELA, E., AND MAIZEL, J. V. (1967) Biochem. Biophys. Res. Commun. 28, tion about such a role first stimulated by 815-820. chemical modification studies by Trown P. T., AND BURGESS, D. R. (1978) and Rabin (33) but continuing in more re- 13. MATSUDAIRA, Anal. Biochem. 87, 386-396. cent articles (34-36). Although Cys-172 R., ANDKOVACS, K. (1974) and Cys-459 in spinach carboxylase appear 14. PENKE, B., FERENCZI, Anal. Biochem. 60, 45-50. to be located in the active-site region based 15. GROSS, E. (1967) in Methods in Enzymology on their reaction with the affinity label N(Hire, C. H. W., ed.), Vol. 11, pp. 238-255, bromoacetylethanolamine phosphate (proAcademic Press, New York. vided the enzyme is deactivated), this does 16. MAHONEY, W. C., ANDHERMODSON, M. A. (1979) not dictate an essentiality in either catalBiochemistry 1S,3810m3814.
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SEQUENCE
88.9 d d
OF
RIBULOSE-P,
CARBOXYLASE
436
HARTMAN
16.1 21.4 29.0 24.1 29.0 *,.* 19.4 8.2
ET
AL.
SEQUENCE
OF
RIBULOSE-P2
437
CARBOXYLASE
8
13.Rd
9 10
8.8
8.8 5.3 4.0 2.6
e