Modification of Rhodospirillum rubrum ribulose bisphosphate carboxylase with pyridoxal phosphate. 2. Stoichiometry and kinetics of inactivation

Biochemistry ◽  
1978 ◽  
Vol 17 (7) ◽  
pp. 1288-1293 ◽  
Author(s):  
William B. Whitman ◽  
F. Robert Tabita
Keyword(s):  
Pyridoxal Phosphate ◽  
Rhodospirillum Rubrum ◽  
Ribulose Bisphosphate Carboxylase ◽  
Ribulose Bisphosphate ◽  
Bisphosphate Carboxylase ◽  
Kinetics Of

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.

scholarly journals Ribulose 1,5-bisphosphate carboxylase. Effect on the catalytic properties of changing methionine-330 to leucine in the Rhodospirillum rubrum enzyme

1986 ◽  
Vol 235 (3) ◽  
pp. 839-846 ◽  
Author(s):  
B E Terzaghi ◽  
W A Laing ◽  
J T Christeller ◽  
G B Petersen ◽  
D F Hill
Keyword(s):  
Rhodospirillum Rubrum ◽  
Oligonucleotide Primer ◽  
Similar Amount ◽  
Ribulose Bisphosphate Carboxylase ◽  
Ribulose Bisphosphate ◽  
Carboxylase Activity ◽  
Bisphosphate Carboxylase ◽  
Ribulose Bisphosphate Carboxylase Oxygenase ◽  
In The Wild ◽  
The Stability

Oligonucleotide-directed mutagenesis of cloned Rhodospirillum rubrum ribulose bisphosphate carboxylase/oxygenase with a synthetic 13mer oligonucleotide primer was used to effect a change at Met-330 to Leu-330. The resultant enzyme was kinetically examined in some detail and the following changes were found. The Km(CO2) increased from 0.16 to 2.35 mM, the Km(ribulose bisphosphate) increased from 0.05 to 1.40 mM for the carboxylase reaction and by a similar amount for the oxygenase reaction. The Ki(O2) increased from 0.17 to 6.00 mM, but the ratio of carboxylase activity to oxygenase activity was scarcely affected by the change in amino acid. The binding of the transition state analogue 2-carboxyribitol 1,5-bisphosphate was reversible in the mutant and essentially irreversible in the wild type enzyme. Inhibition by fructose bisphosphate, competitive with ribulose bisphosphate, was slightly increased in the mutant enzyme. These data suggest that the change of the residue from methionine to leucine decreases the stability of the enediol reaction intermediate.

scholarly journals Ribulose bisphosphate carboxylase/oxygenase in toluene-permeabilized Rhodospirillum rubrum

1983 ◽  
Vol 212 (1) ◽  
pp. 45-54 ◽  
Author(s):  
I Storrø ◽  
B A McFadden
Keyword(s):  
Rhodospirillum Rubrum ◽  
Decay Rates ◽  
Dual Function ◽  
Co2 Removal ◽  
Ribulose Bisphosphate Carboxylase ◽  
Ribulose Bisphosphate ◽  
Carboxylase Activity ◽  
Bisphosphate Carboxylase ◽  
Ribulose Bisphosphate Carboxylase Oxygenase ◽  
The Stability

Toluene-permeabilized Rhodospirillum rubrum cells were used to study activation of and catalysis by the dual-function enzyme ribulose bisphosphate carboxylase/oxygenase. Incubation with CO2 provided as HCO3-, followed by rapid removal of CO2 at 2 degrees C and subsequent incubation at 30 degrees C before assay, enabled a determination of decay rates of the carboxylase and the oxygenase. Half-times at 30 degrees C with 20 mM-Mg2+ were 10.8 and 3.7 min respectively. Additionally, the concentrations of CO2 required for half-maximal activation were 56 and 72 microM for the oxygenase and the carboxylase respectively. After activation and CO2 removal, inactivation of ribulose bisphosphate oxygenase in the presence of 1 mM- or 20mM-Mn2+ was slower than that with the same concentrations of Co2+ or Mg2+. Only the addition of Mg2+ supported ribulose bisphosphate carboxylase activity, as Mn2+, Co2+ and Ni2+ had no effect. A pH increase after activation in the range 6.8-8.0 decreased the stability of the carboxylase but in the range 7.2-8.0 increased the stability of the oxygenase. With regard to catalysis. Km values for ribulose 1,5-bisphosphate4- were 1.5 and 67 microM for the oxygenase and the carboxylase respectively, and 125 microM for O2. Over a broad range of CO2 concentrations in the activation mixture, the pH optima were 7.8 and 8-9.2 for the carboxylase and the oxygenase respectively. The ratio of specific activities was constant (9:1 for the carboxylase/oxygenase) of ribulose bisphosphate carboxylase/oxygenase in toluene-treated Rsp. rubrum. Below concentrations of 10 microM-CO2 in the activation mixture, this ratio increased.

Models of integrated photosynthesis of cells and leaves

1989 ◽  
Vol 323 (1216) ◽  
pp. 357-367 ◽  
Keyword(s):  
Simplified Model ◽  
Ribulose Bisphosphate Carboxylase ◽  
Ribulose Bisphosphate ◽  
Optimal Arrangement ◽  
Bisphosphate Carboxylase ◽  
Ribulose Bisphosphate Carboxylase Oxygenase ◽  
Kinetics Of

The kinetics of ribulose bisphosphate carboxylase-oxygenase (Rubisco) are integrated with the stoichiometry of NADPH consumption and production in a simplified model of C 3 chloroplast photosynthesis. The extension to a leaf is discussed with reference to the gradient of irradiance that is always present within the leaf. The optimal arrangement of photosyntheic capacity is discussed in this context. Attention is then given to the effects of gradients of CO 2 concentration that sometimes occur when stomata close in a heterogeneous fashion.

scholarly journals A model for the kinetics of activation and catalysis of ribulose 1,5-bisphosphate carboxylase

1976 ◽  
Vol 159 (3) ◽  
pp. 563-570 ◽  
Author(s):  
W A Laing ◽  
J T Christeller
Keyword(s):  
Initial Rate ◽  
Equilibrium Constants ◽  
Co2 Concentration ◽  
Time Dependent ◽  
Enzyme Complex ◽  
Ribulose Bisphosphate Carboxylase ◽  
Ribulose Bisphosphate ◽  
Bisphosphate Carboxylase ◽  
Rate Of Reaction ◽  
Kinetics Of

Further evidence for time-dependent interconversions between active and inactive states of ribulose 1,5-bisphosphate carboxylase is presented. It was found that ribulose bisphosphate oxygenase and ribulose bisphosphate carboxylase could be totally inactivated by excluding CO2 and Mg2+ during dialysis of the enzyme at 4 degrees C. When initially inactive enzyme was assayed, the rate of reaction continually increased with time, and the rate was inversely related to the ribulose bisphosphare concentration. The initial rate of fully activated enzyme showed normal Michaelis-Menten kinetics with respect to ribulose bisphosphate (Km = 10muM). Activation was shown to depend on both CO2 and Mg2+ concentrations, with equilibrium constants for activation of about 100muM and 1 mM respectively. In contrast with activation, catalysis appeared to be independent of Mg2+ concentration, but dependent on CO2 concentration, with a Km(CO2) of about 10muM. By studying activation and de-activation of ribulose bisphosphate carboxylase as a function of CO2 and Mg2+ concentrations, the values of the kinetic constants for these actions have been determined. We propose a model for activation and catalysis of ribulose bisphosphate carboxylase: (see book) where E represents free inactive enzyme; complex in parentheses, activated enzyme; R, ribulose bisphosphate; M, Mg2+; C, CO2; P, the product. We propose that ribulose bisphosphate can bind to both the active and inactive forms of the enzyme, and slow inter-conversion between the two states occurs.

The effects of low temperature acclimation of winter rye on catalytic properties of its ribulose bisphosphate carboxylase–oxygenase

1979 ◽  
Vol 57 (7) ◽  
pp. 1036-1041 ◽  
Author(s):  
N. P. A. Huner ◽  
F. D. H. Macdowall
Keyword(s):  
Temperature Acclimation ◽  
Winter Rye ◽  
Ribulose Bisphosphate Carboxylase ◽  
Ribulose Bisphosphate ◽  
Bisphosphate Carboxylase ◽  
Ribulose Bisphosphate Carboxylase Oxygenase ◽  
Secale Cereale L ◽  
Decreasing Ph ◽  
Chemical Groups ◽  
Kinetics Of

A comparison was made of the kinetics of the carboxylation reaction of bicarbonate–magnesium-activated ribulose biphosphate carboxylase–oxygenase purified from cold-hardened and unhardened winter rye (Secale cereale L. cv. Puma). The activity of the (NH4)2SO4− precipitated enzyme from hardened plants was stable at −20 °C for a month, whereas the form from unhardened plants was reversibly cold inactivated. The [Formula: see text] of the unhardened form increased more rapidly with decreasing pH below 8.2, but the estimated pKa of chemical groups associated with the active site was not affected by the cold hardening. The temperature dependencies of the [Formula: see text] of the two forms of the enzyme crossed at 10 °C with the effect that the catalysis of carboxylation by ribulose biphosphate carboxylase–oxygenase from Puma rye was most efficient in the temperature range to which the plants had been adapted.

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