Purification, quaternary structure, composition, and properties of D-ribulose-1,5-bisphosphate carboxylase from Thiobacillus intermedius

Abstract

D-Ribulose-1,5-bisphosphate (RuBP) carboxylase has been purified from glutamate-C02-S203 2-_grown Thiobacillus intermedius by pelleting the enzyme from the high-speed supernatant and by intermediary crystallization followed by sedimentation into a discontinuous 0.2 to 0.8 M sucrose gradient. The enzyme was homogeneous by the criteria of electrophoresis on polyacrylamide gels of several acrylamide concentrations, sedimentation velocity and equilibrium measurements, and electron microscopic observations of negatively stained preparations. The molecular weights of the enzyme determined by sedimentation equilibrium and light-scattering measurements averaged 462,500 + 13,000. The enzyme consisted of closely similar or identical polypeptide chains of a molecular weight of 54,500 5,450 determined by polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulfate. The s°2,, of the enzyme was 18.07S + 0.22. Electron microscopic examination suggested that the octomeric enzyme (inferred from the molecular measurements mentioned) had a cubical structure. The specific activity of the enzyme was 2.76 ,tmol of RuBP-dependent CO2 fixed/min per mg of protein (at pH 8 and 30 C), and the turnover number in terms of moles of CO2 fixed per mole of catalytic site per second was 2.6. The enzyme was stable for 3 months at -20 C and at least 4 weeks at 0 C. The apparent Km for CO2 was 0.75 mM, and Km values for RuBP and Mg2+ were 0.076 and 3.6 mM, respectively. Dialyzed enzyme could be fully reactivated by the addition of 20 mM Mg2+ and partially reactivated by 20 mM Co2+, but Cd2+, Mn2+, Ca2+, and Zn2+ had no effect. The compound 6-phosphogluconate was a linear competitive inhibitor with respect to RuBP when it had been preincubated with enzyme, Mg2+, and HCO3r. D-Ribulose-1,5-bisphosphate carboxylase (RuBPCase; 3-phospho-D-glycerate carboxylyase Ldimerizing], EC 4.1.1.39), the cardinal enzyme of the reductive pentose phosphate pathway, catalyzes the condensation of CO2 and ribulose-1,5-bisphosphate (RuBP) and the cleavage of the intermediate 2-carboxy-3-ketoribitol-1,5-bisphosphate (31) into 3-phosphoglycerate. RuBPCase is present in all photolithotrophic life forms including purple sulfur and nonsulfur bacteria and the green sulfur bacterium Chlorobium limicola f. thiosulfatophilum (37). The distribution, purification, and properties of RuBPCases have been recently reviewed (22,25). RuBPCase is known to be present in several chemolithotrophic bacteria (1,16,19,21,22,23,29,30,32,42), and pure enzymes have been isolated from fructose-grown Hydrogenomonas eutropha, Hydrogenonormas facilis (13,14), and autotrophically grown Thiobacillus novellus (19,20). RuBPCases isolated from plants, algae, and bacteria have been classified into three size categories (22, 25): large (molecular weight 550,000), intermediate (molecular weight 360,000), and small (molecular weight 120,000). With one exception, large RuBPCases isolated from plants, algae, or bacteria consist of two types of subunits, large (molecular weight 51,000 to 58,000) an...

D-ribulose 1,5-bisphosphate carboxylase from Paracoccus denitrificans

“…From the results of sedimentation velocity experiments a sedimentation coefficient of s~o,w--14,0 S (c ~ 0.2.53 mg protein ml -t) was calculated. This value is considerably lower than the average of about 18 S reported for the high molecular weight RuBPCases from various eukaryotic and bacterial sources [11][12][13].…”

d-Ribulose 1,5-bisphosphate carboxylase of the nitrifying bacterium,Nitrobacter agilis

“…agilis is similar to that of T. novellus, A. eutrophus and P. denitrificans [5,6,8,21 ]. Although the molecular weight of RuBPCase from N. agilis is similar to that from T. intermedius it is distinct from the latter which contains only one type of subunit [7]. The significance of the minor component which was slightly smaller than the large subunit of RuBPCase from N. agilis is not clear.…”

“…c The enzyme of T. novellus contains large (L) and small (S) subunits but the relative proportions have not been established [22 ]. genic photosynthetic organisms [2,[5][6][7][8]. Like other large RuBPCases [24], the enzyme ofN.…”

“…approx. 500 000) forms of the carboxylase [3][4][5][6][7][8]. In contrast, nothing is known of the molecular size or subunit composition of the RuBPCase in nitrifying bacteria, though the enzyme has been detected in cell-free preparations ofN.…”

Presence of two subunit types in ribulose 1,5-bisphosphate carboxylase from Thiobacillus intermedius

“…Purohit et al (22) reported that Thiobacillus intermedius, a carboxysome-containing species, possessed an octameric 0-type RuBPCase (Mr 462,500, s20o. = 18.1S).…”