The Experts below are selected from a list of 42 Experts worldwide ranked by ideXlab platform
Helmut Kindl - One of the best experts on this subject based on the ideXlab platform.
-
Sequence analysis of cucumber cotyledon Ribulosebisphosphate Carboxylase/oxygenase activase cDNA.
Biochimica et biophysica acta, 1992Co-Authors: Regina Preisig-müller, Helmut KindlAbstract:Abstract The cDNA encoding Ribulosebisphosphate Carboxylase / oxygenase activase from cotyledons of dark-grown cucumber seedlings has been isolated and sequenced. The 2.0 kilobase pair cDNA encodes a polypeptide with 413 amino acid residues which shows 70% identity with the rubisco activase from green leaves of spinach. The full-length cDNA was used to prepare rubisco activase by in vitro transcription. Subsequent in vitro translation led to the formation of a 46 kDa protein which is the putative precursor of a 42 kDa plastid enzyme.
-
sequence analysis of cucumber cotyledon Ribulosebisphosphate Carboxylase oxygenase activase cdna
Biochimica et Biophysica Acta, 1992Co-Authors: Regina Preisigmuller, Helmut KindlAbstract:Abstract The cDNA encoding Ribulosebisphosphate Carboxylase / oxygenase activase from cotyledons of dark-grown cucumber seedlings has been isolated and sequenced. The 2.0 kilobase pair cDNA encodes a polypeptide with 413 amino acid residues which shows 70% identity with the rubisco activase from green leaves of spinach. The full-length cDNA was used to prepare rubisco activase by in vitro transcription. Subsequent in vitro translation led to the formation of a 46 kDa protein which is the putative precursor of a 42 kDa plastid enzyme.
Alan R. Fersht - One of the best experts on this subject based on the ideXlab platform.
-
Role of phenylalanine-327 in the closure of loop 6 of Ribulosebisphosphate Carboxylase/oxygenase from Rhodospirillum rubrum.
Biochemistry, 1993Co-Authors: Anthony G. Day, Patrick Chène, Alan R. FershtAbstract:Phenylalanine-327 of Ribulosebisphosphate Carboxylase/oxygenase (rubisco) from Rhodospirillum rubrum was mutated to tryptophan, leucine, valine, alanine, and glycine, and was also deleted. The least active mutant, the deletion mutant, exhibits less than 0.5% of the Carboxylase activity of the wild-type enzyme. Steady-state kinetic analysis of F327-->Leu, Val, Ala, Gly mutant enzymes reveals that kcat and the CO2/O2 specificity are unchanged while Km(RuBP) (RuBP = ribulose 1,5-bisphosphate) is drastically increased. The mutant enzyme with the highest value for Km(RuBP),Phe327-->Gly, shows a 165-fold increase (1160 microM compared to 7 microM for the wild-type). The increase in Km(RuBP) suggests an alteration of the ratio kon/koff for RuBP. A longer hydrophobic lateral chain and/or the presence of an aromatic ring in the wild-type enzyme and the Phe327-->Trp mutant enzyme could explain a better packing of loop 6 in the closed conformation and thus a tighter binding of RuBP at the active site.
-
role of phenylalanine 327 in the closure of loop 6 of Ribulosebisphosphate Carboxylase oxygenase from rhodospirillum rubrum
Biochemistry, 1993Co-Authors: Anthony G. Day, Patrick Chène, Alan R. FershtAbstract:Phenylalanine-327 of Ribulosebisphosphate Carboxylase/oxygenase (rubisco) from Rhodospirillum rubrum was mutated to tryptophan, leucine, valine, alanine, and glycine, and was also deleted. The least active mutant, the deletion mutant, exhibits less than 0.5% of the Carboxylase activity of the wild-type enzyme. Steady-state kinetic analysis of F327-->Leu, Val, Ala, Gly mutant enzymes reveals that kcat and the CO2/O2 specificity are unchanged while Km(RuBP) (RuBP = ribulose 1,5-bisphosphate) is drastically increased. The mutant enzyme with the highest value for Km(RuBP),Phe327-->Gly, shows a 165-fold increase (1160 microM compared to 7 microM for the wild-type). The increase in Km(RuBP) suggests an alteration of the ratio kon/koff for RuBP. A longer hydrophobic lateral chain and/or the presence of an aromatic ring in the wild-type enzyme and the Phe327-->Trp mutant enzyme could explain a better packing of loop 6 in the closed conformation and thus a tighter binding of RuBP at the active site.
Regina Preisig-müller - One of the best experts on this subject based on the ideXlab platform.
-
Sequence analysis of cucumber cotyledon Ribulosebisphosphate Carboxylase/oxygenase activase cDNA.
Biochimica et biophysica acta, 1992Co-Authors: Regina Preisig-müller, Helmut KindlAbstract:Abstract The cDNA encoding Ribulosebisphosphate Carboxylase / oxygenase activase from cotyledons of dark-grown cucumber seedlings has been isolated and sequenced. The 2.0 kilobase pair cDNA encodes a polypeptide with 413 amino acid residues which shows 70% identity with the rubisco activase from green leaves of spinach. The full-length cDNA was used to prepare rubisco activase by in vitro transcription. Subsequent in vitro translation led to the formation of a 46 kDa protein which is the putative precursor of a 42 kDa plastid enzyme.
Regina Preisigmuller - One of the best experts on this subject based on the ideXlab platform.
-
sequence analysis of cucumber cotyledon Ribulosebisphosphate Carboxylase oxygenase activase cdna
Biochimica et Biophysica Acta, 1992Co-Authors: Regina Preisigmuller, Helmut KindlAbstract:Abstract The cDNA encoding Ribulosebisphosphate Carboxylase / oxygenase activase from cotyledons of dark-grown cucumber seedlings has been isolated and sequenced. The 2.0 kilobase pair cDNA encodes a polypeptide with 413 amino acid residues which shows 70% identity with the rubisco activase from green leaves of spinach. The full-length cDNA was used to prepare rubisco activase by in vitro transcription. Subsequent in vitro translation led to the formation of a 46 kDa protein which is the putative precursor of a 42 kDa plastid enzyme.
T. J. Andrews - One of the best experts on this subject based on the ideXlab platform.
-
An Improved Method for Measuring the CO2/O2 Specificity of Ribulosebisphosphate Carboxylase-Oxygenase
Functional Plant Biology, 1994Co-Authors: Heather J. Kane, Mk Morell, J Viil, B Entsch, K Paul, T. J. AndrewsAbstract:A simple, but very reproducible, method for measuring the relative specificity of Ribulosebisphosphate Carboxylase-oxygenase for CO2, as opposed to O2, is described. The method uses [1-14C]ribulose bisphosphate as substrate and combines the advantages of supplying both gaseous substrates from the gas phase with HPLC separation of the labelled products. Volumetric or gravimetric accuracy is not required at any stage of the procedure and variations in ionic strength and pH have little effect on the measurements. This leads to excellent reproducibility without the need for normalisation. The average standard deviation was 1.3% of the measured CO2/O2 specificity. Use of very low ribulose bisphosphate concentrations ensures that the gaseous substrates cannot be depleted appreciably during the reaction and enhances the attractiveness of the procedure for measurements with crippled mutant enzymes. The procedure's ability to resolve small differences in relative specificity is demonstrated by its easy detection of the 5% increase in specificity that accompanies substitution of four residues at positions 338-341 of the cyanobacterial large subunit with the analogous higher-plant residues. This resolving power is essential for detecting small differences in the specificities of higher-plant Ribulosebisphosphate Carboxylases which may be the signature of continuing evolutionary refinement.
-
an improved method for measuring the co2 o2 specificity of Ribulosebisphosphate Carboxylase oxygenase
Australian Journal of Plant Physiology, 1994Co-Authors: Heather J. Kane, Mk Morell, J Viil, B Entsch, K Paul, T. J. AndrewsAbstract:A simple, but very reproducible, method for measuring the relative specificity of Ribulosebisphosphate Carboxylase-oxygenase for CO2, as opposed to O2, is described. The method uses [1-14C]ribulose bisphosphate as substrate and combines the advantages of supplying both gaseous substrates from the gas phase with HPLC separation of the labelled products. Volumetric or gravimetric accuracy is not required at any stage of the procedure and variations in ionic strength and pH have little effect on the measurements. This leads to excellent reproducibility without the need for normalisation. The average standard deviation was 1.3% of the measured CO2/O2 specificity. Use of very low ribulose bisphosphate concentrations ensures that the gaseous substrates cannot be depleted appreciably during the reaction and enhances the attractiveness of the procedure for measurements with crippled mutant enzymes. The procedure's ability to resolve small differences in relative specificity is demonstrated by its easy detection of the 5% increase in specificity that accompanies substitution of four residues at positions 338-341 of the cyanobacterial large subunit with the analogous higher-plant residues. This resolving power is essential for detecting small differences in the specificities of higher-plant Ribulosebisphosphate Carboxylases which may be the signature of continuing evolutionary refinement.
-
The source and characteristics of chemiluminescence associated with the oxygenase reaction catalyzed by Mn(2+)-Ribulosebisphosphate Carboxylase.
The Journal of biological chemistry, 1993Co-Authors: R M Lilley, H Riesen, T. J. AndrewsAbstract:We confirm the observation of Mogel and McFadden (Mogel, S.N., and McFadden, B. A. (1990) Biochemistry 29, 8333-8337) that Ribulosebisphosphate Carboxylase/oxygenase (rubisco) exhibits chemiluminescence while catalyzing its oxygenase reaction in the presence of Mn2+. However, our results with the spinach and Rhodospirillum rubrum enzymes differ markedly in the following respects. 1) Chemiluminescence intensity was directly proportional to enzyme concentration and behaved as if representing the rate of oxygenase catalysis. 2) The wavelength spectrum peaked at about 770 nm and extended beyond 810 nm. This seems inconsistent with chemiluminescence generated by simultaneous decay of pairs of singlet O2 molecules. It is consistent with manganese(II) luminescence and we discuss its possible sources. The time course of chemiluminescence (resolution, 0.25 s) was distinctively different for spinach and R. rubrum enzymes during the initial 5 s of catalysis, with the bacterial enzyme exhibiting a pronounced initial "burst." Chemiluminescence by the spinach enzyme responded to substrate concentrations in a manner consistent with known oxygenase properties, exhibiting Michaelis-Menten kinetics with ribulose-1,5-bisphosphate (Km 400 nM). Chemiluminescence required carbamylated enzyme with Mn2+ bound at the active site (activation energy, -57.1 KJ.mol-1). As an indicator of oxygenase activity, chemiluminescence represents an improvement over oxygen electrode measurements in response time and sensitivity by factors of at least 100.
-
Pyruvate is a by-product of catalysis by Ribulosebisphosphate Carboxylase/oxygenase.
The Journal of biological chemistry, 1991Co-Authors: T. J. Andrews, H J KaneAbstract:Pyruvate is a minor product of the reaction catalyzed by Ribulosebisphosphate Carboxylase/oxygenase from spinach leaves. Labeled pyruvate was detected, in addition to the major labeled product, 3-phosphoglycerate, when 14CO2 was the substrate. Pyruvate production was also measured spectrophotometrically in the presence of lactate dehydrogenase and NADH. The Km for CO2 of the pyruvate-producing activity was 12.5 microM, similar to the CO2 affinity of the 3-phosphoglycerate-producing activity. No pyruvate was detected by the coupled assay when ribulose 1,5-bisphosphate was replaced by 3-phosphoglycerate or when the Carboxylase was inhibited by the reaction-intermediate analog, 2'-carboxyarabinitol 1,5-bisphosphate. Therefore, pyruvate was not being produced from 3-phosphoglycerate by contaminant enzymes. The ratio of pyruvate produced to ribulose bisphosphate consumed at 25 degrees C was 0.7%, and this ratio was not altered by varying pH or CO2 concentration or by substituting Mn2+ for Mg2+ as the catalytically essential metal. The ratio increased with increasing temperature. Ribulose-bisphosphate Carboxylases from the cyanobacterium Synechococcus PCC 6301 and the bacterium Rhodospirillum rubrum also catalyzed pyruvate formation and to the same extent as the spinach enzyme. When the reaction was carried out in 2H2O, the spinach Carboxylase increased the proportion of its product partitioned to pyruvate to 2.2%. These observations provide evidence that the C-2 carbanion form of 3-phosphoglycerate is an intermediate in the catalytic sequence of ribulose-bisphosphate Carboxylase. Pyruvate is formed by beta elimination of a phosphate ion from a small portion of this intermediate.
-
pyruvate is a by product of catalysis by Ribulosebisphosphate Carboxylase oxygenase
Journal of Biological Chemistry, 1991Co-Authors: T. J. Andrews, H J KaneAbstract:Pyruvate is a minor product of the reaction catalyzed by Ribulosebisphosphate Carboxylase/oxygenase from spinach leaves. Labeled pyruvate was detected, in addition to the major labeled product, 3-phosphoglycerate, when 14CO2 was the substrate. Pyruvate production was also measured spectrophotometrically in the presence of lactate dehydrogenase and NADH. The Km for CO2 of the pyruvate-producing activity was 12.5 microM, similar to the CO2 affinity of the 3-phosphoglycerate-producing activity. No pyruvate was detected by the coupled assay when ribulose 1,5-bisphosphate was replaced by 3-phosphoglycerate or when the Carboxylase was inhibited by the reaction-intermediate analog, 2'-carboxyarabinitol 1,5-bisphosphate. Therefore, pyruvate was not being produced from 3-phosphoglycerate by contaminant enzymes. The ratio of pyruvate produced to ribulose bisphosphate consumed at 25 degrees C was 0.7%, and this ratio was not altered by varying pH or CO2 concentration or by substituting Mn2+ for Mg2+ as the catalytically essential metal. The ratio increased with increasing temperature. Ribulose-bisphosphate Carboxylases from the cyanobacterium Synechococcus PCC 6301 and the bacterium Rhodospirillum rubrum also catalyzed pyruvate formation and to the same extent as the spinach enzyme. When the reaction was carried out in 2H2O, the spinach Carboxylase increased the proportion of its product partitioned to pyruvate to 2.2%. These observations provide evidence that the C-2 carbanion form of 3-phosphoglycerate is an intermediate in the catalytic sequence of ribulose-bisphosphate Carboxylase. Pyruvate is formed by beta elimination of a phosphate ion from a small portion of this intermediate.
