It has been hypothesized that components of enzymatic pathways might organize into intracellular assemblies to improve their catalytic efficiency or lead to coordinate regulation. Accordingly, de novo purine biosynthesis enzymes may form a purinosome in the absence of purines, and a punctate intracellular body has been identified as the purinosome. We investigated the mechanism by which human de novo purine biosynthetic enzymes might be organized into purinosomes, especially under differing cellular conditions. Irregardless of the activity of bodies formed by endogenous enzymes, we demonstrate that intracellular bodies formed by transiently transfected, fluorescently tagged human purine biosynthesis proteins are best explained as protein aggregation.
The carboxylase activity of ribulose 1,5-bisphosphate carboxylase/oxygenase (RuBPC/O) decreased when an anaerobic culture of RhodospiriUum rubrum was exposed to atmospheric levels of oxygen. From 70 to 80% of the activity was lost within 12 to 24 h. Inactivation was apparent when the enzyme was assayed in situ (in whole cells) and when activity was measured in dialyzed crude extracts. The quantity of enzyme protein, as estimated from sodium dodecyl sulfate-polyacrylamide gels or as quantified immunologically, did not decrease within 24 h of exposure to air. Following extended exposure to aerobic conditions (48 to 72 h), degradation of enzyme occurred. These results indicate that the inactivation of RuBPC/O in R. rubrum may be due to an alteration or modification of the preformed enzyme, followed by eventual degradation of the inactive enzyme. When shifted back to anaerobic conditions (under an argon atmosphere), the RuBPC/O activity increased rapidly. This increase appeared to be due to de novo synthesis of enzyme. The increase in activity was not observed when the culture was maintained in the dark or in the absence of a suitable carbon source. Thus, the oxygen-mediated inactivation of RuBPC/O appeared to be due to some form of irreversible modification. The cloned R. rubrum RuBPC/O gene, expressed in Escherichia coli, yielded functional enzyme that was not affected by oxygen, indicating that inactivation in R. rubrum is mediated by a gene product(s) not found in E. coli.Ribulose 1,5-bisphosphate (RuBP) carboxylase/oxygenase (RuBPC/O) catalyzes the first reaction of both the reductive and oxidative photosynthetic carbon cycles (10). In the absence of oxygen, RuBPC/O catalyzes the carboxylation and cleavage of RuBP, resulting in the formation of two molecules of 3-phosphoglyceric acid. In the presence of oxygen, RuBPC/O catalyzes the oxygenolysis of RuBP, resulting in the formation of 1 molecule of 3-phosphoglyceric acid and 1 molecule of phosphoglycolate. Usually the phosphoglycolate formed is further metabolized via the glycolate pathway, resulting in the wasteful loss of carbon and nitrogen. The oxygenase function of the enzyme therefore results ultimately in a futile cycle, with loss of CO2 and enormous energy expenditure (5). The ability of RuBPC/O to catalyze these two distinct reactions necessitates careful metabolic regulation of enzyme activity. Rhodospirillum rubrum, like all members of the family Rhodospirillaceae, synthesizes high levels of RuBPC/O when grown under anaerobic photosynthetic conditions with butyrate or hydrogen as the electron donor (1,13,15,16,18). In the present investigation, it is shown that RuBPC/O is metabolically regulated in R. rubrum following exposure to oxygen. The evidence suggests that inactivation occurs by some form of alteration or modification of the enzyme when cells are exposed to atmospheric levels of oxygen. The physiological significance of this response is evident and may be related to the need to inactivate RuBPC/O in order to limit the wasteful loss of carbon a...
Ribulose 1,5-bisphosphate (RuBP) carboxylase/oxygenase (RuBPC/0) was inactivated in crude extracts of Rhodospirilum rubrum under atmospheric levels of oxygen; no inactivation occurred under an atmosphere of argon. RuBP carboxylase activity did not decrease in dialyzed extracts, indicating that a dialyzable factor was required for inactivation. The inactivation was inhibited by catalase. Purified RuBPC/0 is relatively oxygen stable, as no loss of activity was observed after 4 h under an oxygen atmosphere. The aerobic inactivation catalyzed by endogenous factors in crude extracts was mimicked by using a model system containing purified enzyme, ascorbate, and FeSO4 or FeCl3. Dithiothreitol was found to substitute for ascorbate in the model system. Preincubation of the purified enzyme with RuBP led to enhanced inactivation, whereas Mg2+ and HC03-significantly protected against inactivation. Unlike the inactivation catalyzed by endogenous factors from extracts of R. rubrum, inactivation in the model system was not inhibited by catalase. It is proposed that ascorbate and iron, in the presence of oxygen, generate a reactive oxygen species which reacts with a residue at the activation site, rendering the enzyme inactive.Evidence has been presented that aerobic inactivation of ribulose 1,5-bisphosphate (RuBP) carboxylase/oxygenase (RuBPC/O) in Rhodospirillum rubrum proceeds by a twostep process: the enzyme is first inactivated by an alteration or modification, and then the inactive protein is proteolytically degraded (2). This is very similar to well-described systems, such as glutamine synthetase (GS) and glutamine phosphoribosylpyrophosphate aminotransferase, in which oxidative inactivation precedes the actual degradation of the modified protein (11,21). With regard to GS, inactivation may be mediated by a variety of enzymatic [cytochrome P-450, glucose oxidase, horseradish peroxidase, NAD(P)H oxidase, and xanthine oxidase] and nonenzymatic [ascorbic acid, dihydroxyfumaric acid, and NAD(P)H plus menadione] systems (14). Inactivation was found to result in the loss of 1 of 16 histidine residues, with the subsequent formation of 1 carbonyl group per GS subunit (8). Levine has suggested that a mixed-function oxidation system generates a reactive oxygen species which reacts with a histidine residue to introduce a carbonyl moiety at the active site of the enzyme (9, 10). The oxidatively modified form of GS then becomes increasingly vulnerable to intracellular and exogenous proteases (16)(17)(18).A number of other procaryotic and eucaryotic enzymes are also susceptible to oxidative modification. Pyruvate kinase, creatine kinase, lactate dehydrogenase (14), 3-phosphoglycerate kinase (3), superoxide dismutase (5), and tyrosinase (7) are all susceptible to oxidative inactivation. Thus, loss of catalytic activity due to oxidative modification appears to be a common method of enzymatic regulation. In this study, evidence is presented that RuBPC/O is also susceptible to oxidative modification in vitro.
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