Abstract
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.
Highlights
Pyruvate is a minor productof the reaction catalyzed ase (EC4.1.1.39) is fundamental to almost all foromf sphotoby ribulosebisphosphate carboxylase/oxygenase from or chemolithotrophic life
The ratio as I1 and 111) hasprovenstable enough to be isolatedin of pyruvate producedtoribulosebisphosphateconquantity, and its reactions in solution and on the active site sumed a t 2 5“C was 0.7%,and this ratio was not altehraevde been characterized (Schloss and Lorimer, 1982; Pierce by varying pH or COzconcentration orby substituting et al, 1986; Lorimer et al, 1986;Andrews and Lorimer,1987)
When recently by observations thatisolated ribulose-Pncarboxylase the reaction was carried out i‘nHzO, the spinach car- slowly epimerizes ribulose-P2 toproduce the potent inhibitor, boxylase increased the proportion of its product par- D-xyhlOSe 1,5-bisphosphate,leading to progressive inhibition titioned to pyruvate to 2.2%
Summary
P, became limiting, and it eventually ceased when ribuloseP, was exhausted. The extentof the reaction corresponded to Materials-Ribulose-Pc, arboxylase was purifiedfrom spinach less than 1%of the ribulose-P, initiallypresent(TableI). Catalysis was initiated by adding spinachribuloseP, carboxylase (final concentration of catalytic sites, 2.1 p ~w)hich had been preactivated in the presence of 19 mM MgCI, and 10 mM system used in the pyruvateassay. All of these data confirm that pyruvate must be produced as an integral part of the reaction mechanismof ribulose-P, carboxylase. Ribulose-P2 concentrations were measured spectrophotometrically usingan assay similar to that describedfor P-glycerate in the legend of Table I, reactions (Table Io)r from their initial rates (data not shown). The ratio was approximately 0.7% and was remarkably unaffected by varying conditions (Table I)F. or spinach ribulose-P2 carboxylase, varying pH between 6.4 and 9.1 was without effect, as except that 10 mMHCO: and 20 pg/ml spinach ribulose-P2 carbox- was varying theHCO, concentration between 0.5 and 10 mM ylase were present
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