Comments on the structure and function of the large subunit of the enzyme ribulose bisphosphate carboxylase-oxygenase

Poulsen, Carsten Stig

doi:10.1007/bf02906502

Cited by 33 publications

(17 citation statements)

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“…Each of the peptides derived from RbuP2 carboxylase from wheat, tobacco, and muskmelon contained blocked N termini; no phenylthiohydantoin derivatives were released after five cycles of Edman degradative sequencing. Amino acid composition analyses of these peptides yielded similar results to the peptide derived from spinach RbuP2 carboxylase ( Tryptic peptides were partially purified as described (6) and applied to an octyldecyl silica reversephase column (10-,um particle size, 4.6 x 220 mm Aquapore column, Brownlee Lab, Santa Clara, CA) equilibrated with aqueous 0.07% trifluoroacetic acid. After Met-Ser-Pro-Gl n-Thr-Gl u-Thr-Lys…”

Section: Resultsmentioning

confidence: 90%

Post-translational modifications in the large subunit of ribulose bisphosphate carboxylase/oxygenase.

Houtz

Stults

Mulligan

et al. 1989

Proc. Natl. Acad. Sci. U.S.A.

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Two adjacent N-terminal tryptic peptides of the large subunit of ribulose bisphosphate carboxylase/oxygenase [3-phospho-D-glycerate carboxy-lyase (dimerizing), EC 4.1.1.39] from spinach, wheat, tobacco, and muskmelon were removed by limited tryptic proteolysis. Characterization by peptide sequencing, amino acid composition, and tandem mass spectrometry revealed that the N-terminal residue from the large subunit of the enzyme from each plant species was acetylated proline. The sequence of the penultimate N-terminal tryptic peptide from the large subunit of the spinach and wheat enzyme was consistent with previous primary structure determinations. However, the penultimate N-terminal peptide from the large subunit of both the tobacco and muskmelon enzymes, while identical, differed from the corresponding peptide from spinach and wheat by containing a trimethyllysyl residue at position 14. Thus, tryptic proteolysis occurred at lysine-18 rather than lysine-14 as with the spinach and wheat enzymes. A comparison of the DNA sequences for the large subunit of ribulose bisphosphate carboxylase/oxygenase indicates that the N terminus has been post-translationally processed by removal of methionine-1 and serine-2 followed by acetylation of proline-3. In addition, for the enzyme from tobacco and muskmelon a third post-translational modification occurs at lysine-14 in the form of NE-trimethylation.Ribulose bisphosphate carboxylase/oxygenase [RbuP2 carboxylase, 3-phospho-D-glycerate carboxy-lyase (dimerizing), EC 4.1.1.39] is a hexadecameric plant protein with eight large subunits (LSs) and eight small subunits (SSs) (for a recent review, see ref.

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Section: Resultsmentioning

confidence: 90%

Post-translational modifications in the large subunit of ribulose bisphosphate carboxylase/oxygenase.

Houtz

Stults

Mulligan

et al. 1989

Proc. Natl. Acad. Sci. U.S.A.

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“…Early reports indicate that the LS of RbuP2 carboxylase was synthesized as a precursor and posttranslationally processed to a form 1-2 kDa smaller (26). Other amino-terminal determinations have indicated that Ala-15 in barley (27) or both Ala-15 and Thr-5 in tobacco (28) were the amino-terminal residues. The amino-terminal tryptic peptide sequence of peptide 2 ('thr:glx:pro:lys=2:2:11) ala ser val gly phe lys met ser pro gln thr glu thr lys ala ser val glu phe lys ala gly val lys ... of the wheat LS has been reported to contain Ser-2, but apparently lacked Met-1 (20).…”

Section: Discussionmentioning

confidence: 99%

Reaction-intermediate analogue binding by ribulose bisphosphate carboxylase/oxygenase causes specific changes in proteolytic sensitivity: the amino-terminal residue of the large subunit is acetylated proline.

Mulligan

Houtz

Tolbert

1988

Proc. Natl. Acad. Sci. U.S.A.

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Trypsin rapidly inactivated the catalytic activities of spinach ribulose bisphosphate carboxylase/oxygenase [3-phospho-D-glycerate carboxy-lyase (dimerizing), EC 4.1.1.39], but the stoichiometry of binding of the reactionintermediate analogue carboxyarabinitol bisphosphate was only slightly reduced after proteolysis. Electrophoretic analysis indicated that several forms of the large subunit were generated during proteolysis but that the small subunit was resistant. Three tryptic peptides were isolated and characterized after digestion of the activated enzyme; the trypticsensitive sites were identified at Lys-8, Lys-14, and Lys-466 of the large subunit. Ribulose bisphosphate carboxylase/oxygenase [RbuP2 carboxylase, 3-phospho-D-glycerate carboxy-lyase (dimerizing), EC 4.1.1.39] catalyzes both the carboxylation and oxygenation of RbuP2, which represent the first committed reactions of both the reductive and oxidative photosynthetic carbon cycles (1). Although considerable detail is known about the mechanism of the carboxylation reaction (2, 3), little is known about the dynamics of the protein during activation, 2'-C-carboxyarabinitol 1,5-bisphosphate (CABP) binding, or catalysis. Activation of RbuP2 carboxylase by CO2 and Mg2 + results in conformational changes as measured by changes in sedimentation velocity (4), circular dichroism (5), and reactivity with active-site probes (6,7). Binding of CABP also results in conformational changes in the protein as documented by changes in absorbance spectroscopy (8), electrophoretic mobility (9), and x-ray crystallography patterns (10). With the exception of active-site derivatization, these studies have not provided specific molecular information, such as regions or individual amino acid residues that change in conformation or reactivity.In this report proteolysis has been utilized to probe the structure and function of RbuP2 carboxylase. Susceptibility of native proteins to proteases may increase or decrease due to ligand-induced conformational changes (11). A specific peptide bond at Lys-466 in the carboxyl-terminal region of RbuP2 carboxylase radically changed sensitivity to trypsin after CABP binding. In addition, characterization of the peptides released from spinach RbuP2 carboxylase showed that the amino-terminal amino acid Pro-3 is acetylated and that the amino acid sequence predicted from the DNA sequence is in error at residue 12. A preliminary report of these results has been published (12). MATERIALS AND METHODSEnzyme Preparation, Assay, and Treatment with Trypsin.RbuP2 carboxylase was purified to homogeneity from spinach leaves, activated, and assayed as described (13,14).RbuP2 carboxylase was activated at 2 mg/ml and exposed to trypsin (10 ,ug/ml) at 30°C. The initial proteolysis was terminated by addition of 150 ,ug of trypsin inhibitor per ,g of trypsin before assaying enzyme activity. The stoichiometry of CABP bound by RbuP2 carboxylase was determined as described (13).Electrophoresis. Discontinuous NaDodSO4/polyacrylamide gels were prepared wit...

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“…In most cases the enzyme is composed of two types ofsubunits, eight identical catalytic, large subunits (LS) of 52,000-56,000 MW each and eight small subunits (SS) of 12,000-15,000 MW a piece (21). The role of the small subunit in the assembled enzyme is still enigmatic.…”

Section: Introductionmentioning

confidence: 99%

“…The nucleotide sequences of the genes for the subunits have revealed that the small subunit amino acid sequences differ widely among species (5) whereas the amino acid sequences of the large subunits are more strongly conserved (6,7,21,28). Conserved domains in the small subunit are of interest in elucidating the mechanisms of transportation of the small subunit into the chloroplasts (5) and assembly of the enzyme (3,29).…”

Section: Introductionmentioning

confidence: 99%

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Two mRNA species differing by 258 nucleotides at the 5′ end are formed from the barley chloroplast rbcL gene

Poulsen

1984

Carlsberg Res. Commun.

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The region of the barley chloroplast genome encoding the 5' third of the rbeL gene has been subcloned in M 13 vectors and sequenced by the dideoxy chain termination method. Some of the recombinant M 13 clones have been used as a template for the synthesis of highly radioactive single stranded DNA probes. These are specifically hybridizing to short regions at the 5' end of the two transcripts, which were previously found to be encoded by the rbcL region in barley cpDNA. The two transcripts differ in size by more than 200 nucleotides, are encoded by the same strand and contain the full coding capacity for the large subunit of ribulose bisphosphate carboxylase-oxygenase. The short transcript is predominant in dark grown seedlings. Of two probes employed in this study one hybridized only to the long and the other to both rbcL mRNA species. Thereby it was possible to quantitate the rbeL m RNAs by hybridization with the probes and subsequent S l-nuclease digestion of the single stranded molecules. I found this mRNA to be aproximately 1.38% of total plastid RNA (excluding the small rRNAs and the tRNAs) in dark grown seedlings and 2. 20% in greened seedlings. The two probes were also used in primer extension experiments using the two transcripts as template. After hybridizing the probe to the mRNA reverse transcription was performed in the presence of cold deoxynucleotides. The reverse transcribed strands were separated on sequencing gels. The probe which acted as a specific primer for the short transcript was extended by 29 nucleotides, and the probe specific for the longer transcript was extended by 91 nucleotides. The end point of the latter transcript was only found with template RNA from plastids of greened barley seedlings. Thus it was shown that the 5" ends of the two transcripts begin at positions -58 and -316 from the ATG triplet of the rbcL reading frame. A comparison of the sequences upstream from the ATG codon of the rbeL gene of barley with those of maize, spinach and tobacco revealed completely conserved prokaryote type transcription promotors in front of the 5' end of the long transcript, while no such promotors are evident close to the 5' end point of the short transcript. The two monoeot species differ from the dicot species by an insertion of about 140 nucleotides in the mRNA leader sequence.Abbreviations: atpB = the gene for ATP synthetase CF, subunit ~; bp(s) = basepair(s); cpDNA = chloroplast DNA; dsDNA = double stranded DNA; kb(s) = kilobase(s); kbp(s) = kilobasepair(s); LS = large subunit; psbA = gene for a 32,000 dalton photosystem II protein; rbeL = gene for the RuBPCase-Oase LS; RF = replicative form; RuBPCase-Oase = ribulose bisphosphate carboxylase-oxygenase; ssDNA = single stranded DNA.Springer-Verlag

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Comments on the structure and function of the large subunit of the enzyme ribulose bisphosphate carboxylase-oxygenase

Cited by 33 publications

References 57 publications

Post-translational modifications in the large subunit of ribulose bisphosphate carboxylase/oxygenase.

Post-translational modifications in the large subunit of ribulose bisphosphate carboxylase/oxygenase.

Reaction-intermediate analogue binding by ribulose bisphosphate carboxylase/oxygenase causes specific changes in proteolytic sensitivity: the amino-terminal residue of the large subunit is acetylated proline.

Two mRNA species differing by 258 nucleotides at the 5′ end are formed from the barley chloroplast rbcL gene

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