FASE III: Comunicación de Resultados

In a soluble extract of photosynthetic eukaryotic cells, up to 65% of the protein can be accounted for by the Rubisco enzyme, and thus Rubisco has been claimed to be 'the most abundant protein on earth' (Ellis, 1979). The reason for its abundance appears to be the fact that the enzyme has a low turnover number (to.). At saturating substrate concentrations one molecule of Rubisco fixes approximately 1000 molecules of CO2 per minute (compare e.g.

P

galactosidase, t.o.=12 500 and carbonic anhydrase, t.o.=3 6000 000 (Lehninger, 1970)).

In the atmospheric concentrations o f CO2 the turnover number o f the Rubisco enzyme is

about 200; thus the organism has to synthesize a large number of molecules to meet the required demand for carbon fixation. A low specificity (about 2|j.mol.min~l.mg‘l) has been determined for Rubisco purified from both prokaryotic and eukaryotic cells. It appears that even prokaryotes, which are under severe selection pressure, have not managed to evolve a more efficient Rubisco enzyme (Ellis & Gatenby, 1984). The abundance of Rubisco and its low specific activity make it an ideal candidate to study the molecular mechanism of catalysis which may elucidate why Rubisco is such an inefficient catalyst. The remainder o f this section discusses the processes involved in catalysis in detail.

Pon et al. (1963) were the first to recognize that Rubisco must be activated before it catalyzes either the carboxylation or the oxygenation of ribulose-1.5-bisphosphate. Early studies showed that in both isolated intact chloroplasts and leaves the Km(CC>2) was similar to the CO2 concentration (10 pM) found in aqueous solution in equilibrium with air,

whereas the Km(C02) for the Rubisco purified from Spinacia oleracea was approximately

ten times higher (reviewed by Walker, 1973). Bahr & Jensen (1974) identified a form of the enzyme which has a high affinity for CO2, in hypotonic preparations of lysed chloroplasts. This form of Rubisco was shown to be stabilized by CO2 (or bicarbonate) and Mg2+ ¡ons

(Bahr & Jensen, 1974; Andrews et al., 1975).Thus CO2 acts both as an activator and a

substrate. The activation of the Rubisco enzyme is an ordered process (Lorimer et al., 1976)

in which CO2 reacts with an e-lysyl amino group to fotm a carbamate, thereby generating a negatively-charged carbonyl group which provides a site for interaction with the positively- charged Mg2+ ion. Thus the activation of the enzyme can be decribes as follows (Lorimer & Miziorko, 1980), where E represents the Rubisco enzyme:-

H+

E-Lys-NH3-t* E-Lys-NH2 + CO2— E-Lys-NH-COC>- + Mg2+~~ E-Lys-NH-C02'.Mg2+ H+

(inactive) (active)

The rate-limiting step appears to be the formation of the carbamate which occurs at Lys residue 201 in the spinach enzyme (Lorimer, 1981) and at Lys residue 91 in the

R.rubrum dimeric enzyme (Donnelly et al„ 1983). Since the activation of Rubisco in vivo is

promoted by light this raises the question how the observations made in vitro can be

interpreted for the in vivo situation. It appears that during illumination the concentration of

CO2 probably changes little, whereas the Mg2+ ion concentration and the pH in the stroma of the chloroplast increase (reviewed by Buchanan, 1980), and this could thus promote the

carbamylation of the e-amino group. However the Kact(CC>2) for Rubisco determined in

vitro is approximately 25-30 (J.M (Lorimer et al„ 1976; Jordan & Ogren,1981), whereas

almost fully activated Rubisco has been observed in vivo at high light intensity in air,

conditions under which the CO2 concentration in the chloroplast is approximately 10 pM

(Perchorowicz & Jensen, 1983; Salvucci et al„ 1986). These observations suggest that an

addditional factor which allows maximum activation of the Rubisco enzyme is present in

vivo. With the aid of a mutant of Arabidopsis thaliana which was defective in Rubisco

activation (Somerville eta l., 1982), the additional factor was identified as a soluble protein

and was called Rubisco activase (Salvucci et al„ 1985). The Rubisco activase has been

purified from Spinacia oleracea and activates purified Rubisco in an ATP-dependent fashion

(Streusand & Portis, 1987) indicating that ATP hydrolysis may be required.

The reactions catalyzed by the activated Rubisco enzyme have been studied in great

detail (reviewed by Lorimer et al., 1986; Gutteridge & Gatenby, 1987). The fate of the

various atoms involved in the formation o f 3-phosphoglycerate and 2-phosphoglycolate from ribulose-1.5-bisphosphate and CO2 and 0 2 respectively is summarized in Figure 3. The first step is the formation of the enediol form of ribulose bisphosphate (product of reaction 1, Fig. 3A) which requires only the activated Rubisco enzyme. It is the enediol form to which either CO2 or 0 2 binds. Once the substrate is bound the RuBP is converted into either two molecules of 3-phosphoglycerate or one molecule o f 3-phosphoglycerate and one molecule of 2-phosphoglycolate respectively. The recent determination of the quaternary

structure of the dimeric Rubisco from R.rubrum at 1.7 A resolution has provided detailed

information about the mode of binding of CO2, the Mg^+ ion and the transtition state

Figure 3. Rubisco catalysis and su b strate binding

The overall reaction which is catalyzed by the Rubisco enzyme can be disseted into five steps (Panel A): 1. enolization, involving the protonation of C4 of ribulose-1.5-bisphosphate; 2. either initiation of carboxylation or oxygenation by electrophilic addition of gaseous carbon dioxide (CO2) oxygen (02) respectively; 3. hydration of the C3 carbonyl by H2O; 4. carbon-carbon cleavage to yield a molecule of 3-phosphoglycerate (lower molecule) from the bottom of the six carbon bisphosphate intermediate, and either one molecule of C-2 carbanion derivative or one molecule of 2-phosphoglycolate in the carboxylase and oxygenase reaction respectively; 5. protonation of the C-2 carbanion derivative on the enzyme to give the second molecule of 3-phosphoglycerate in the carboxylase reaction. This

diagram was designed based on data by Lorimer et al. (1986) and Gutteridge & Gatenby

(1987).

Panel B shows a schematic diagram of interactions between the Rubisco protein side chains

and the metal atom (red) of the activated enzyme from Spinacia oleracea. The bound

transition state analogue 2-carboxy-D-arabinitol-1.5-bisphosphate (2CABP) is shown in

yellow. This diagram was taken from a recent paper by Andersson et al. (1989).

A

CHaOPOj* c=o H-i- m C HjO P Oj' C HjO P Oj' H - C - O H I C HjO P Oj' CH7OP03* C H ,O P O ^ - C — C O * CHjOPOa" H O - C - O - O ' I H O - C - O H - I H - C - O H I ch7o po3j‘ 0 C H70 P 0 3 I HO —C — C 0 7 c h7o p o3j' c h2o p oJ- ho- c_I -co;₇ H 0 c h_I 7o p o:‘₃

A

2- C H jO P O ]'

B

35/2

analogue 2-carboxy-D-arabinitol-1.5-bisphosphate (2CABP) to amino acid residues at the

active site of the enzyme from R.rubrum and S.oleracea (Andersson et al., 1989). The

schematic diagram representing the different interactions is shown in Figure 3B.

Despite all the information available regarding the reactions catalyzed by Rubisco it is still unknown what determines the efficiency with which carbon is fixed. Since all Rubisco enzymes examined exhibit both carboxylase and oxygenase activities, it has been argued that the oxygenase activity is an inevitable consequence of the active site chemistry (reviewed by Ellis & Gatenby ,1984); however the balance between the carboxylation and oxygenation has

been shown to vary. The ratio of carboxylation to oxygenation has been defined by Laing et

al. (1974) as follows:-

vc/vo = (VcKo/VoKc ) ([CO2MQ2])

where vc and vo are the velocities of carboxylation and oxygenation respectively, Vc and Vo the maximal velocities o f the two reactions, and Kc and Ko the Michaelis constants for CC>2 and O2. The substrate specificity (VcKo/VoKc) determines the rates of the two reactions at any given CO2 and O2 concentration; a high specificity value indicate a greater specificity for CO2. Jordan & Ogren (1981; 1983) determined the substrate specificity factor for a variety

of organisms and observed a low value for the form II Rubisco from R.rubrum (15),

whereas the substrate specificity factor for the form I Rubisco from cyanobacteria and higher plants was approximately 48 and 80 respectively. Jordan and Ogren concluded that modifications increasing the carboxylation efficiency had occurred during evolution. The observation that the enzyme activity is lost from the hexadecameric Rubisco enzyme upon removal of the small subunits indicates their absolute requirement for catalysis.

1-2.3 SUBUNIT SYNTHESIS

In eukaryotes the Rubisco enzyme is found in the stromal compartment of the chloroplast. Since the first report in 1962 that chloroplasts contain all the components

required to carry out protein synthesis (reviewed by Kirk & Tilney-Bassett, 1978), much research has been carried out to determine which proteins are synthesized in this plant organelle (reviewed by Ellis, 1981). The use of specific inhibitors such as chloramphenicol and cycloheximide, which inhibit chloroplast 70S and cytosolic 80S ribosomes respectively,

allowed the uncoupling of the two protein-synthesizing systems (Criddle et al., 1970). The

inhibition of chloroplast protein synthesis by chloramphenicol was shown to result in an inhibition of the synthesis of Rubisco large subunits in greening barley shoots, whereas the synthesis of the Rubisco small subunits was inhibited by cycloheximide. These observations

led Criddle et al. (1970) to conclude that the Rubisco large subunits are synthesized inside

the chloroplast while the small subunits are synthesized by cytosolic ribosomes. Uncoupling

of the subunits using specific inhibitors has also been reported for Pisum sativum shoots

(Cashmore, 1976), Nicotiana tabacum protoplasts (Hirai & Wildman, 1977) and Glycine

max cells (Barraclough & Ellis, 1979).

In young leaves of several cereal seedlings such as Secale ceraele L (rye), the

formation of 70S but not 80S ribosomes is inhibited at an elevated temperature between 32 °C and 34 °C (Feierabend & Schrader-Reichhardt, 1976). This phenomenon was utilized to demonstrate that plants grown at the non-permissive temperature fail to synthesize Rubisco large subunits, but do synthesize Rubisco small subunits (Feierabend & Wildner, 1978). The demonstration that the large subunit is inherited in a non-Mendelian fashion in

Nicotiana tabacum (Sakano et al., 1974) and in Chlamydomonas reinhardii (Spreitzer & Mets, 1980) provided genetic evidence that the Rubisco large subunit is not nuclear- encoded, and thus is probably encoded in the chloroplast genome.

Direct evidence that the Rubisco large subunits are synthesized on chloroplast ribosomes came from experiments with isolated intact chloroplasts. Blair & Ellis (1973)

were the first to demonstrate that in intact chloroplast isolated from Pisum sativum leaves,

protein synthesis can be driven by light energy if chloroplasts are incubated in an appropriate medium containing radiolabelled amino acids. The major polypeptide synthesized inside these isolated chloroplasts was identified as the large subunit of Rubisco. Synthesis of the

Euglena gracilis, Sorghum vulgare and Acetabularia (reviewed by Ellis, 1981). Whereas these above observations provide good evidence that the large subunit is synthesized inside the chloroplast they only suggest that the large subunit may be encoded by the chloroplast genome. Direct evidence for this idea was obtained when chloroplast DNA, rather than

RNA, was isolated from Zea mays leaves, transcribed in vitro by E.coli RNA polymerase,

and the transcripts subsequently translated in a cell-free rabbit reticulocyte lysate. Among the large number of polypeptides synthesized the Rubisco large subunit was identified as the

most abundant (Coen et al., 1977). From these initial observations it was concluded that the

Rubisco large subunit is encoded by the chloroplast genome and synthesized within the chloroplast.

A precursor of the large subunit with an apparent M r of approximately 2000 larger

than the mature polypeptide was identified when total RNA from Spinacia oleracea

chloroplasts was translated in an E.coli cell-free system (Landridge, 1981). Treatment of the

precursor with a soluble chloroplast extract converts it to its mature size indicating the presence of a processing activity. In support of possible post-translational processing of the large subunit is the observed discrepancy between the determined amino acid sequence of the

mature large subunit from Hordeum vulgare (Poulsen, 1981), Triticum aestivum and

Sorghum vulgare (Miziorko & Lorimer,1983) and the deduced amino acid sequence

(McIntosh et al., 1980; Zurawski et al., 1981; Dron et al., 1982; Shinozaki & Sugiura,

1982), where the latter contains 14 additional amino acid residues at the aminoterminal end.

Whether the processing occurs in vivo or in vitro during protein purification remains to be

established. It is noted that in Nicotiana tabacum the determined (Amiri et al., 1984) and the

deduced (Shinozaki & Sugiura, 1982) amino acid sequence of the large subunit are identical at the aminoterminus.

The Rubisco small subunit is a product of cytosolic protein synthesis since its

synthesis is inhibited in vivo by cycloheximide but not by chloramphenicaol (Criddle et al.,

1970; Cashmore, 1976; Barraclough & Ellis, 1979). Genetic analysis in Nicotiana tabacum

showed a Mendelian inheritanic for the Rubisco small subunit (Kawashima & Wildman, 1972), provided the first evidence that the small subunit is nuclear-encoded. Furthermore,

the small subunit mRNA was shown to be translated in vitro on free cytosolic ribosomes to give a precursor polypeptide with an apparent Mr about 6000 larger than the mature

polypeptide (Dobberstein et al., 1977; Cashmore et al., 1978; Highfield & Ellis, 1978),

whereas transcripts coding for the small subunit precursor have been identified in both the

nuclear and cytosolic compartments of leaf cells from Pisum sativum (Smith & Ellis, 1981;

Gallagher & Ellis, 1982).

The synthesis of Rubisco in prokaryotes is less complex due to the lack o f cellular compartments. In cyanobacteria the small subunit is synthesized as the mature polypeptide, and there is no evidence for the synthesis of a large subunit precursor.

The observation that the synthesis of the Rubisco subunits in eukaryotes occurs in two different cellular compartments, whereas the assembled Rubisco enzyme is found only inside the chloroplast implies that the small subunit precursor is transported accross the chloroplast envelope prior to assembly (see Section 1.2.4) and suggests that a coordinate regulation of Rubisco subunit synthesis may exist (see Section 1.2.5).

In document Auditoría de gestión a la Cooperativa de Transportes Macuchi, de la ciudad Mana, provincia de Cotopaxi, periodo 2014 (página 116-178)