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Query: UNIPROT:P00790 (
PGA
)
2,475
document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)
Ribulose
-diphosphate carboxylase from Thiobacillus novellus has been purified to hemogeneity as observed by polyacrylamide gel electrophoresis and U.V. light observation during sedimentation velocity analysis. The optimum pH for the enzyme with Tris-HCl buffers was about 8.2. Concentrations of this buffer in excess of 80 mM were inhibitory. The apparent Km for RuDP was about 14.8 muM with a Hill value of 1.5, for HCO3- the apparent Km was about 11.7 mM with an n value of 1.18 and for Mg2+ about 0.61 mM. The enzyme was specific for this cation. Relatively high concentrations of either Hg2+ or pCMB were required before significant inhibition was observed. Activity declined slowly during a 4-hr incubation period in either 3.0 M or 8.0 M urea. Incubation for 12 hrs resulted in complete loss of activity which was not prevented by 10 mM Mg2+ and was not reversed by dialysis and subsequent addition of 10 mM cysteine. Polyacrylamide gel electrophoresis revealed a loss of the major band and the appearance of 2 new bands. SDS polyacrylamide gel electrophoresis gave an average M.W. of 73500 +/- 2500 for the slower moving band and 12250 +/- 2500 for the faster moving. However, incubation in urea for up to 40 hrs revealed a decrease in the M.W. of the slower moving band to about 60000. The Ea for the enzyme was calculated to be about 18.85 kcal mole-1, with the possibility of a "break" between 40 and 50 degrees C. The Q10 was 3.07 between 20 and 30 degrees C whereas between 30 to 40 degrees C it was 3.31. Only phosphorylated compounds caused significant inhibition of enzyme activity. They included ADP, FDP, F6P, G6P, PEP, 6PG, 2-
PGA
, R1P, R5P, and Ru5p.
...
PMID:Properties and regulation of ribulose diphosphate carboxylase from Thiobacillus novellus. 24 94
An assay was developed for simultaneous kinetic analysis of the activities of the bifunctional plant enzyme ribulose 1,5-bisphosphate carboxylase/oxygenase [EC 4.1.1.39]. [1-(14)C,5-(3)H]
Ribulose
1,5-bisphosphate (RuBP) was used as the labeled substrate. Tritium enrichment of the doubly labeled 3-phosphoglycerate (3-PGA) product, common to both enzyme activities, may be used to calculate V(c)/V(o) ratios from the expression A/(B-A) where A and B represent the (3)H/(14)C isotope ratios of doubly labeled RuBP and 3-
PGA
, and V(c) and V(o) represent the activities of carboxylase and oxygenase, respectively. Doubly labeled substrate was synthesized from [2-(14)C]glucose and [6-(3)H]glucose using the enzymes of the pentose phosphate pathway coupled with phosphoribulokinase.The kinetic properties of a commercial preparation of fully activated spinach carboxylase were studied under approximated physiological conditions of 20% O(2) (252 micromolar), 295 mul/l CO(2) (10 micromolar), 25 C, and pH 8.19. The V(c)/V(o) ratio was, within experimental error, constant at 30 seconds and 1 minute. This double label assay method may be used to calculate V(c)/V(o) ratios for the Laing-Ogren-Hageman equation, V(c)/V(o) = (V(c)K(o)/V(o)K(c)) ([CO(2)]/[O(2)]) where V(c) and V(o) represent V(max), and K(c) and K(o) represent Michaelis constants for the carboxylase and oxygenase activities, respectively.
...
PMID:Simultaneous kinetic analysis of ribulose 1,5-bisphosphate carboxylase/oxygenase activities. 1666 Dec 14
Ribulose
1,5-bisphosphate carboxylase-oxygenase (Rubisco) is the primary carbon-fixing enzyme in photosynthesis, fixing CO
2
to a 5-carbon sugar, RuBP, in a series of five reactions. However, it also catalyzes an oxygenase reaction by O
2
addition to the same enolized RuBP substrate in an analogous reaction series in the same active site, producing a waste product and loss of photosynthetic efficiency. Starting from RuBP, the reactions are enolization to the enediolate form, addition of CO
2
or O
2
to form the carboxy or peroxo adduct, hydration to form a gemdiolate, scission of the C2-C3 bond of the original RuBP, and stereospecific or nonstereospecific protonation to form two molecules of the 3-carbon
PGA
product, or one molecule of
PGA
, one of 2-carbon PG (waste product), and one water molecule. Reducing the loss of efficiency from the oxygenase reaction is an attractive means to increase crop productivity. However, lack of understanding of key aspects of the catalytic mechanisms for both the carboxylase and oxygenase reactions, particularly those involving proton exchanges and roles of water molecules, has stymied efforts at re-engineering Rubisco to reduce losses from the oxygenation reaction. As the stable form of molecular oxygen is the triplet biradical state (
3
O
2
), its reaction with near-universal singlet-state molecules is formally spin forbidden. Although in oxygenase enzymes,
3
O
2
activation is usually achieved by one-electron transfers using transition-metal ions or organic cofactors, recently, cofactor-less oxygenases in which the substrate itself is the source of the electron for
3
O
2
activation have been identified, but in all such cases an aromatic ring stabilizes the substrate's negative charge. Here we present the first large-scale Kohn-Sham density functional theory study of the reaction mechanism of the Rubisco oxygenase pathway. First, we show that the enediolate substrate complexed to Mg
2+
and its ligands extends the region for charge delocalization and stabilization of its negative charge to allow formation of a caged biradical enediolate-O
2
complex. Thus, Rubisco is a unique type of oxygenase without precedent in the literature. Second, for the O
2
addition to proceed to the singlet peroxo-adduct intermediate, the system must undergo an intersystem crossing. We found that the presence of protonated LYS334 is required to stabilize this intermediate and that both factors (strongly stabilized anion and protonated LYS334) facilitate a barrier-less activation of
3
O
2
. This finding supports our recent proposal that deoxygenation, that is, reversal of gas binding, is possible. Third, as neither CO
2
nor O
2
binds to the enzyme, our findings support the proposal from our recent carboxylase study that the observed K
C
or K
O
(Michaelis-Menten constants) in the steady-state kinetics reflect the respective adducts, carboxy or peroxo. Fourth, after computing hydration pathways with water addition both syn and anti to C3, we found, in contrast to the results of our carboxylation study indicating anti addition, that in the oxygenation reaction only syn-hydration is capable of producing a stable gemdiolate that facilitates the rate-limiting C2-C3 bond scission to final products. Fifth, we propose that an excess proton we previously found was required in the carboxylation reaction for activating the C2-C3 bond scission is utilized in the oxygenation reaction for the required elimination of a water molecule. In summary, despite its oxygenase handicap, Rubisco's success in directing 75% of its substrate through the carboxylation pathway can be considered impressively effective. Although native C3 Rubiscos are in a fix with unwanted activity of
3
O
2
hampering its primary carboxylase function, mechanistic differences presented here with findings in our recent carboxylase study for both the gas-addition and subsequent reactions provide some clues as to how creative Rubisco re-engineering may offer a solution to reducing the oxygenase activity.
...
PMID:Mechanism of Oxygenase-Pathway Reactions Catalyzed by Rubisco from Large-Scale Kohn-Sham Density Functional Calculations. 3084 2
