Phosphoenolpyruvate carboxylase from the crassulacean plant <i>Bryophyllum fedtschenkoi</i> Hamet et Perrier. Purification, molecular and kinetic properties

Jones, R. Forbes; Wilkins, Malcolm B.; Coggins, John R.; Fewson, Charles A.; Malcolm, Alan D. B.

doi:10.1042/bj1750391

Cited by 69 publications

(42 citation statements)

References 36 publications

(54 reference statements)

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“…The estimated monomer mol wt of alfalfa nodule PEPC of 97,000 based on migration in SDS polyacrylamide gels is similar to monomer subunit weights of maize leaf PEPC (100,000) and Bryophyllum fedtschenkoi (100,000), both sources in which the enzyme is believed to be a tetramer of identical monomers (6,17). Mol wt estimations have not been reported for either soybean or lupine nodule PEPC.…”

Section: Methodsmentioning

confidence: 65%

Alfalfa Root Nodule Carbon Dioxide Fixation

Vance¹,

Stade²

1984

Plant Physiol.

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A nonphotosynthetic phosphoenolpyruvate carboxylase (EC 4.1.131) was partially purified from the cytosol of root nodules of alfalfa. The enzyme was purified 86-fold by ammonium sulfate fractionation, DEAEcellulose, hydroxylapatite chromatography, and reactive agarose with a final yield of 32%. The enzyme exhibited a pH optimum of 7.5 with apparent K,. values for phosphoenolpyruvate and magnesium of 210 and 100 micromolar, respectively. Two isozymes were resolved by nondenaturing polyacrylamide disc gel electrophoresis. Subsequent electrophoresis of these isozymes in a second dimension by sodium dodecyl sulfate slab gel electrophoresis yielded identical protein patterms for the isozymes with one major protein band at molecular weight 97,000. Malate and AMP were slightly inhibitory (about 20%) to the partially purified enzyme. Phosphoenolpyruvate carboxylase comprised approximately 1 to 2% of the total soluble protein in actively N2-fixing alfalfa nodules.Recent studies have demonstrated that nodules of several legume species actively fix CO2 via the enzyme PEPC2 (EC 4.1.1.31) and that PEPC may increase nodule carbon use efficiency by recycling a portion of the CO2 lost through nodule respiration (1,2,7,20). In alfalfa, nodule CO2 fixation is highly correlated with active N2-fixation and has been shown to contribute up to 25% of the carbon required for the assimilation of symbiotically derived N (20). Both in vitro PEPC activity and in vivo CO2 fixation activity in alfalfa nodules decreased following applications of NO3-(20).Although PEPC has been purified and characterized from nodules of the annual legumes soybean and lupine, regulation of the enzyme activity is not well understood (1, 13). Soybean nodule PEPC activity appears to be regulated by PEP and HCO3 concentrations (13

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Section: Methodsmentioning

confidence: 65%

Alfalfa Root Nodule Carbon Dioxide Fixation

Vance¹,

Stade²

1984

Plant Physiol.

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“…Several workers have reported plant PEPc enzymes ofdifferent sizes (2,5,14). We This dimeric form is inhibited by the malate present and although capable of being activated by G-6-P, the concentration of this effector also shows a diurnal change and its concentration decreases (2).…”

Section: Methodsmentioning

confidence: 99%

Diurnal Regulation of Phosphoenolpyruvate Carboxylase from Crassula

Wu¹,

Wedding²

1985

Plant Physiol.

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Phosphoenolpyruvate carboxylase appears to be located in or associated with the chloroplasts of Crassula. As has been found with this enzyme in other CAM plants, a crude extract of leaves gathered during darkness and rapidly assayed for phosphoenolpyruvate carboxylase (PEPc) activity is relatively insensitive to inhibition by malate. After illumination begins, the PEPc activity becomes progressively more sensitive to malate. This enzyme also shows a diurnal change in activation by glucose-6-phosphate, with the enzyme from dark leaves more strongly activated than that from leaves in the light.When the enzyme is partially purified in the presence of malate, the characteristic sensitivity of the day leaf enzyme is largely retained. Partial purification of the enzyme from dark leaves results in a small increase in sensitivity to malate inhibition.Partially purified enzyme is found by polyacrylamide gel electrophoresis analysis to have two bands of PEPc activity. In enzymes from dark leaves, the slower moving band predominates, but in the light, the faster moving band is preponderant. Both of these bands are shown by sodium dodecyl sulfate-polyacrylamide gel electrophoresis to be composed of the same subunit of 103,000 daltons.The enzyme partially purified from night leaves has a pH optimum of 5.6, and is relatively insensitive to malate inhibition over the range from pH 4.5 to 8. The enzyme from day leaves has a pH optimum of 6.6 and is strongly inhibited by malate at pH values below 7, but becomes insensitive at higher pH values. Activation by glucose-6-phosphate is of the mixed type for the day form of the enzyme, causing both a decreased K,,, for phosphoenolpyruvate and an increased V.,,,,, but the night, or insensitive, form shows only an increase in V,,., in response to glucose-6-phosphate.The daily cycle of nonphotosynthetic CO2 fixation followed by decarboxylation to produce CO2 within leaves closed to gas ' Such a system depends on the sequential use of the product of one set of reactions as the substrate of the next. For example, pyruvate produced by the malic enzyme can be converted to PEP by puruvate kinase to become the substrate of PEP carboxylase. The malate produced by PEPc and malate dehydrogenase from this PEP is the substrate of malic enzyme. Such a sequence of reactions is certain to become a futile cycle unless the components of the system are under rigid control. The central role of malate in such a system is apparent. It has been shown that high concentrations of malate such as those which accumulate during the night in most CAM plants are capable, particularly in conjunction with a low pH, ofconverting the NAD malic enzyme from a low activity dimeric form to a high activity tetramer (21). Malate has long been postulated as a means of regulating the activity PEPc (10), and studies with crude extracts of CAM plant leaves have shown (11,19,22) that during the day the enzyme is strongly inhibited by malate, while after short periods of darkness it is relatively insensitive to inhibition...

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“…The interaction between subunits seems to play an important role in the kinetic and regulatory properties of PEP carboxylase, Some evidence from cross-linking experiments [62], titration of sulfbydryl groups [32] and dissociation by chemical mo~ficatio~ j3Of indicates that an asymmetric ~r~gement of the subunits could exist under some conditions. Resonance energy transfer measurements in the enzyme modified with fluorescent reagents seem to support this hypothesis (Wagner, R., Gonzalez, D.H., PodestB, F-E, and Andreo, C.S., unpublished),…”

Section: Structurementioning

confidence: 99%

“…However, the mechanism of this regulation is still not clear. CAM PEP carboxylase is also activated by glucose 6-phosphate [43] and inhibited by L-malate [37,43,62,76]. The inhibition by L-malate is competitive and shows a cooperative effect [37,62].…”

Section: Cam Plantsmentioning

confidence: 99%