An Improved Method for Measuring the CO2/O2 Specificity of Ribulosebisphosphate Carboxylase-Oxygenase

1994 ◽  
Vol 21 (4) ◽  
pp. 449 ◽  
Author(s):  
HJ Kane ◽  
J Viil ◽  
B Entsch ◽  
K Paul ◽  
MK Morell ◽  
...  
Keyword(s):  
Large Subunit ◽  
Resolving Power ◽  
Plant Residues ◽  
Ribulose Bisphosphate ◽  
Higher Plant ◽  
Average Standard Deviation ◽  
Relative Specificity ◽  
Reproducible Method ◽  
Ribulosebisphosphate Carboxylase ◽  
Excellent Reproducibility

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.

Limitation of the Rate of Ribulosebisphosphate Carboxylase Activation by Carbamylation and Ribulosebisphosphate Carboxylase Activase Activity: Development and Tests of a Mechanistic Model

1996 ◽  
Vol 23 (2) ◽  
pp. 141 ◽  
Author(s):  
IE Woodrow ◽  
ME Kelly ◽  
KA Mott
Keyword(s):  
Mechanistic Model ◽  
Large Subunit ◽  
Intercellular Co2 Concentration ◽  
Rubisco Activase ◽  
Photon Flux ◽  
Photon Flux Density ◽  
Bisphosphate Carboxylase ◽  
Ribulosebisphosphate Carboxylase ◽  
Kinetics Of ◽  
Rubisco Activation

A mechanistically-based model of light-mediated activation of ribulose-1,5-bisphosphate carboxylase/oxygenase (Rubisco) is developed. The model describes the kinetics of Rubisco activation following a relatively rapid increase in photon flux density (PPFD) from an initially low level. Underlying the model is the assumption that there are two slow processes that could potentially limit the rate of light-mediated Rubisco activation. These processes are the addition of the activator CO2 to the large subunit of Rubisco, which is accompanied by a conformational change in the enzyme (carbamylation), and activase-mediated removal of ribulose 1,5-bisphosphate from the inactive form of the enzyme. The contribution of these slow processes to the overall activation kinetics of Rubisco was resolved by measuring Rubisco activation in whole spinach leaves using non-steady-state CO2 exchange. It was found that when the change in PPFD was relatively small and a correspondingly small proportion of the Rubisco pool was activated, the kinetics of activation were highly sensitive to the intercellular CO2 concentration (ci). The apparent rate constant for activation under these conditions was found to be similar to that for the carbamylation of purified spinach Rubisco. When the change in PPFD and the proportion of Rubisco activated was relatively large, however, the kinetics of Rubisco activation were almost completely CO2 insensitive and were consistent with those of an enzyme-catalysed reaction. It is suggested that (1) CO2-insensitive activation reflects the operation of Rubisco activase and (2) the increasing CO2 sensitivity seen as the change in PPFD decreases reflects a transition to limitation by carbamylation.

Comparison of the structure and function of ribulosebisphosphate carboxylase–oxygenase from a cold-hardy and nonhardy potato species

1981 ◽  
Vol 59 (4) ◽  
pp. 280-289 ◽  
Author(s):  
Norman P. A. Huner ◽  
Jiwan P. Palta ◽  
Paul H. Li ◽  
John V. Carter
Keyword(s):  
Large Subunit ◽  
Structure And Function ◽  
Sulfhydryl Groups ◽  
Relative Mass ◽  
Nitrobenzoic Acid ◽  
Significant Difference ◽  
Ribulosebisphosphate Carboxylase ◽  
Cold Hardy ◽  
And Function ◽  
Kinetics Of

A comparison of ribulosebisphosphate carboxylase–oxygenase from the leaves of the non-acclimated, cold-hardy species, Solanum commersonii, and the nonacclimated, nonhardy species, Solanum tuberosum showed that this enzyme from the two species differed in structure and function. The results of sulfhydryl group titration with 5,5′-dithiobis(2-nitrobenzoic acid) indicated that the kinetics of titration and the number of accessible sulfhydryl groups in the native enzymes were different. After 30 min, the enzyme from the hardy species had 1.7 times fewer sulfhydryl groups titrated than that from the nonhardy species. In the presence of 1% (w/v) sodium dodecyl sulfate, the total number of sulfhydryl groups titratable with 5,5′-dithiobis-(2-nitrobenzoic acid) was the same for both species. However, this denaturant had a differential effect on the kinetics of titration with 5,5′-dithiobis(2-nitrobenzoic acid). Both enzymes had a native molecular weight of about 550 000. The quaternary structures of the two enzymes were similar with the presence of large and small subunits of 54 000 and 14 000, respectively. However, there was more polypeptide of 108 000 – 110 000 present in preparations of the enzyme from S. tuberosum than from S. commersonii. This polypeptide is an apparent dimer of the large subunit on a relative mass basis. The large subunit of the enzyme from S. tuberosum was more sensitive to the absence of reducing agent and was more sensitive to freezing and thawing than the large subunit of the enzyme from S. commersonii. Catalytic properties of both enzymes at 5 and 25 °C indicated no significant difference in the [Formula: see text] at either temperature. However, the Vmax at 5 °C for the enzyme from S. commersonii was 35% higher than that of the enzyme from S. tuberosum. In contrast, the Vmax at 25 °C for the enzyme of the hardy species was 250% lower than that of the enzyme from the nonhardy species.

scholarly journals A kinetic study of ribulose bisphosphate carboxylase from the photosynthetic bacterium Rhodospirillum rubrum

1978 ◽  
Vol 173 (2) ◽  
pp. 467-473 ◽  
Author(s):  
J T Christeller ◽  
W A Laing
Keyword(s):  
Rhodospirillum Rubrum ◽  
Photosynthetic Bacterium ◽  
Active Enzyme ◽  
Ribulose Bisphosphate Carboxylase ◽  
Ribulose Bisphosphate ◽  
Higher Plant ◽  
Activation Kinetics ◽  
Bisphosphate Carboxylase ◽  
Proposed Model ◽  
Kinetics Of

The activation kinetics of purified Rhodospirillum rubrum ribulose bisphosphate carboxylase were analysed. The equilibrium constant for activation by CO(2) was 600 micron and that for activation by Mg2+ was 90 micron, and the second-order activation constant for the reaction of CO(2) with inactive enzyme (k+1) was 0.25×10(-3)min-1 . micron-1. The latter value was considerably lower than the k+1 for higher-plant enzyme (7×10(-3)-10×10(-3)min-1 . micron-1). 6-Phosphogluconate had little effect on the active enzyme, and increased the extent of activation of inactive enzyme. Ribulose bisphosphate also increased the extent of activation and did not inhibit the rate of activation. This effect might have been mediated through a reaction product, 2-phosphoglycolic acid, which also stimulated the extent of activation of the enzyme. The active enzyme had a Km (CO2) of 300 micron-CO2, a Km (ribulose bisphosphate) of 11–18 micron-ribulose bisphosphate and a Vmax. of up to 3 mumol/min per mg of protein. These data are discussed in relation to the proposed model for activation and catalysis of ribulose bisphosphate carboxylase.

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