Kinetic Analysis of effectors of phosphoenolpyruvate carboxylase from Bryophyllum fedtschenkoi

Pays, Annette; Jones, R. Forbes; Wilkins, Malcolm B.; Fewson, Charles A.; Malcolm, Alan D. B.

doi:10.1016/0005-2744(80)90176-x

Cited by 26 publications

(6 citation statements)

References 20 publications

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“…Chymostatin, Mr marker proteins for SDS/polyacrylamide-gel electrophoresis, Hepes and D-isoascorbic acid were obtained from Sigma Chemical Co., Poole, Dorset, U.K. Ferritin was from Pharmacia, Milton Keynes, Bucks., U.K. Enzymes were from Boehringer Corp. (London) Ltd., Lewes, Sussex, U.K. The sources of other materials were as given previously [2,8,[20][21][22].…”

Section: Experimental Materialsmentioning

confidence: 99%

Purification, oligomerization state and malate sensitivity of maize leaf phosphoenolpyruvate carboxylase

McNaughton

Fewson

Wilkins

et al. 1989

Biochemical Journal

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A method was developed for the purification of phosphoenolpyruvate carboxylase from darkened maize leaves so that the enzyme retained its sensitivity to inhibition by malate. The procedure depended on the prevention of proteolysis by the inclusion of chymostatin in the buffers used during the purification. The purified enzyme was indistinguishable from that in crude extracts as judged by native polyacrylamide-gel electrophoresis. SDS/polyacrylamide-gel electrophoresis followed by immunoblotting, and Superose 6 gel filtration. Gel-filtration studies showed that the purified enzyme and the enzyme in extracts of darkened or illuminated leaves showed a concentration-dependent dissociation of tetrameric into dimeric forms. Purified phosphoenolpyruvate carboxylase and enzyme in crude extracts from darkened leaves were equally sensitive to inhibition by malate (Ki approx. 0.30 mM) under conditions where it existed in the tetrameric or dimeric forms, but the enzyme in crude extracts from illuminated leaves was less sensitive to malate inhibition (Ki approx. 0.95 mM) whether it was present as a tetramer or as a dimer. It is concluded that changes in the oligomerization state of phosphoenolpyruvate carboxylase are not directly involved in its regulation by light.

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Section: Experimental Materialsmentioning

confidence: 99%

Purification, oligomerization state and malate sensitivity of maize leaf phosphoenolpyruvate carboxylase

McNaughton

Fewson

Wilkins

et al. 1989

Biochemical Journal

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“…Kinetic and regulatory properties of PEPC in CAM plants have been studied mainly in partially purified preparations [t 1,13] ; in one case the enzyme was purified to homogeneity [8] and kinetic parameters for substrate and malate were studied [18]. Apparently, all the above studies refer to the active state of the enzyme, which might differ substantially from the alleged in vivo state in the light.…”

Section: Introductionmentioning

confidence: 99%

Changes in properties of phosphoenolpyruvate carboxylase from the CAM plant Sedum praealtum D.C. upon dark/light transition and their stabilization by glycerol

Manetas

1982

Photosynth Res

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A prenounced decrease in phosphoenolpyruvate earboxylase (PEPC) activity is observed upon dark/light transition in Sedum praealtum D.C., only when glycerol is included in the extraction medium. If glycerol is omitted, the activity extracted in light is initially low, but soon reaches night levels. The stabilization of the light-induced form of the enzyme by glycerol, in crude or desalted extracts, made it possible to study its kinetic properties in comparison to those of the dark form. The behaviour towards substrate (PEP) changes from hyperbolic (dark) to sigmoid (light), S0.5 is increased and the enzymic activity becomes more sensitive to malate inhibition. Quite different activity/pH profiles are also obtained for the two forms of PEPC.It is inferred that the in vivo regulation of PEPC in CAM is effected by a concerted action of light, malate and pH shifting.

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“…G6P appears to be a mixed type activator for the day form of the enzyme, causing both decreased Km for PEP and an increased Vmax, but the night form shows only an increase in Vmax in presence of G6P (25, 28 (1,16,19,27). PEP carboxylase in CAM plants is a regulatory enzyme which is activated by G6P and inhibited by malate, the end product of the carboxylation pathway in vivo (20,25,28). This enzyme exists in both a day form and a night form, which have different kinetic properties with regard to the substrates and the effec-…”

Section: Introductionmentioning

confidence: 99%

“…The enzyme is found in bacteria, algae, and plants (1,23), and is of special importance in plants which utilize C4 pathway of photosynthetic carbon fixation and in plants which exhibit CAM (1,16,19,27). PEP carboxylase in CAM plants is a regulatory enzyme which is activated by G6P and inhibited by malate, the end product of the carboxylation pathway in vivo (20,25,28 …”

mentioning

confidence: 99%

Effect of Diethylpyrocarbonate on the Allosteric Properties of Phosphoenolpyruvate Carboxylase from Crassula argentea

Taghizadeh¹,

Jacoby²,

Grover³

1991

Plant Physiol.

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Phosphoenolpyruvate carboxylase from the Crassulacean acid metabolism plant Crassula argentea was substantially desensitized to the effects of regulatory ligands by treatment with diethylpyrocarbonate, a reagent which selectively modifies histidyl residues. Desensitization of the enzyme to the inhibitor malate and the activator glucose 6-phosphate was accompanied by the appearance of a peak in the ultraviolet difference spectrum at 240 nanometers, indicating the formation of ethoxyformylhistidyl derivatives. Hydroxylamine reversed part of the spectral change under native conditions, and almost all of the change under denaturing conditions, but failed to restore sensitivity to effectors. The pH profiles of desensitization to malate and glucose 6-phosphate indicated the involvement of groups on the enzyme with pKa values of 6.8 and 6.4, respectively. Under denaturing conditions, a total of 15 histidine residues per subunit were modified by diethylpyrocarbonate, whereas for the native enzyme nine histidines were modified per subunit. Effector desensitization occurs after the modification of two to three histidyl residues per subunit. The presence of malate reduced the apparent rate constant for desensitization by 60%, suggesting that the modification occurred at the malate binding site. Diethylpyrocarbonate treatment also eliminated the kinetic lag caused by malate. Glucose 6-phosphate did not protect the enzyme against diethylpyrocarbonate-induced desensitization.tors. G6P appears to be a mixed type activator for the day form of the enzyme, causing both decreased Km for PEP and an increased Vmax, but the night form shows only an increase in Vmax in presence of G6P (25, 28 (1,16,19,27). PEP carboxylase in CAM plants is a regulatory enzyme which is activated by G6P and inhibited by malate, the end product of the carboxylation pathway in vivo (20,25,28). This enzyme exists in both a day form and a night form, which have different kinetic properties with regard to the substrates and the effec-

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Kinetic Analysis of effectors of phosphoenolpyruvate carboxylase from Bryophyllum fedtschenkoi

Cited by 26 publications

References 20 publications

Purification, oligomerization state and malate sensitivity of maize leaf phosphoenolpyruvate carboxylase

Purification, oligomerization state and malate sensitivity of maize leaf phosphoenolpyruvate carboxylase

Changes in properties of phosphoenolpyruvate carboxylase from the CAM plant Sedum praealtum D.C. upon dark/light transition and their stabilization by glycerol

Effect of Diethylpyrocarbonate on the Allosteric Properties of Phosphoenolpyruvate Carboxylase from Crassula argentea

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