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- All Subjects: Proteolytic enzymes
- Creators: Wachter, Rebekka M.
Description
ABSTRACT
The catalytic chaperone of Rubisco is AAA+ protein Rubisco activase (Rca), which hydrolyzes ATP and thus undergoes conformational change, helping in reactivating Rubisco. Rca reactivates Rubisco plausibly by removing its C- terminal tail from the opening of its active site thus releasing the inhibitor, a sugar phosphate molecule. Rubisco and Rca are regulated by the stromal environment, which includes the ATP/ADP ratio, Mg2+ concentration, redox potential etc. Here the mechanistic regulation of tobacco β-Rca was studied using steady state enzyme kinetics in terms of product inhibition, Mg2+ activation, cooperativity and asymmetry. A continuous Pi measurement assay was developed, and using this assay catalytic parameters were obtained, such as kcat 20.6 ± 6.5 min-1 ( n = 9) and KM 0.113 ± 0.033 mM (n = 4). A Mg2+ induced increase of substrate affinity in Rca was observed, where the KM changes from 0.452 mM to 0.069 mM, with the changing of free Mg2+ concentration from 0.1 mM to 10 mM. Fitting the catalytic efficiency as a function free Mg2+ concentration by use of a binding model gave a Hill coefficient of 2.2, which indicates a secondary magnesium binding site on the enzyme. A 8.4 fold increase of catalytic efficiency with increasing magnesium from 0.1 mM to 6.5 mM suggests a significant Mg2+ induced regulation of Rca. Moderate product inhibition was observed in inhibition study (Ki = 0. 063 ± 0.018 mM). A positive cooperativity (nH = 2.1) in ATP hydrolysis between two subunits was observed in the presence of 0.132 mM ADP, but not in the absence of ADP. This indicated the presence of two different classes of subunits, suggesting an asymmetric model for the enzyme. Inhibited Rubisco (ER) up to 20 μM concentration did not affect ATPase activity, in line with previous reports. The concentration dependent correlation of Rca activity (tobacco β-Rca) and oligomerization (cotton β-Rca) suggested that the dimer maybe the most active oligomeric species. A nucleotide induced thermal stabilization of Rca was observed, where ADP is more stabilizing than ATP in the absence of Mg2+. Mg2+ has a small destabilizing effect alone and in presence of the ADP, but a stabilizing effect in presence of ATP. The ligand induced thermal stability was similar for cotton and tobacco β-Rca.
The catalytic chaperone of Rubisco is AAA+ protein Rubisco activase (Rca), which hydrolyzes ATP and thus undergoes conformational change, helping in reactivating Rubisco. Rca reactivates Rubisco plausibly by removing its C- terminal tail from the opening of its active site thus releasing the inhibitor, a sugar phosphate molecule. Rubisco and Rca are regulated by the stromal environment, which includes the ATP/ADP ratio, Mg2+ concentration, redox potential etc. Here the mechanistic regulation of tobacco β-Rca was studied using steady state enzyme kinetics in terms of product inhibition, Mg2+ activation, cooperativity and asymmetry. A continuous Pi measurement assay was developed, and using this assay catalytic parameters were obtained, such as kcat 20.6 ± 6.5 min-1 ( n = 9) and KM 0.113 ± 0.033 mM (n = 4). A Mg2+ induced increase of substrate affinity in Rca was observed, where the KM changes from 0.452 mM to 0.069 mM, with the changing of free Mg2+ concentration from 0.1 mM to 10 mM. Fitting the catalytic efficiency as a function free Mg2+ concentration by use of a binding model gave a Hill coefficient of 2.2, which indicates a secondary magnesium binding site on the enzyme. A 8.4 fold increase of catalytic efficiency with increasing magnesium from 0.1 mM to 6.5 mM suggests a significant Mg2+ induced regulation of Rca. Moderate product inhibition was observed in inhibition study (Ki = 0. 063 ± 0.018 mM). A positive cooperativity (nH = 2.1) in ATP hydrolysis between two subunits was observed in the presence of 0.132 mM ADP, but not in the absence of ADP. This indicated the presence of two different classes of subunits, suggesting an asymmetric model for the enzyme. Inhibited Rubisco (ER) up to 20 μM concentration did not affect ATPase activity, in line with previous reports. The concentration dependent correlation of Rca activity (tobacco β-Rca) and oligomerization (cotton β-Rca) suggested that the dimer maybe the most active oligomeric species. A nucleotide induced thermal stabilization of Rca was observed, where ADP is more stabilizing than ATP in the absence of Mg2+. Mg2+ has a small destabilizing effect alone and in presence of the ADP, but a stabilizing effect in presence of ATP. The ligand induced thermal stability was similar for cotton and tobacco β-Rca.
ContributorsHazra, Suratna (Author) / Wachter, Rebekka M. (Thesis advisor) / Fromme, Petra (Committee member) / Frasch, Wayne D (Committee member) / Arizona State University (Publisher)
Created2015
Description
The primary carbon fixing enzyme Rubisco maintains its activity through release of trapped inhibitors by Rubisco activase (Rca). Very little is known about the interaction, but binding has been proposed to be weak and transient. Extensive effort was made to develop Förster resonance energy transfer (FRET) based assays to understand the physical interaction between Rubisco and Rca, as well as understand subunit exchange in Rca.
Preparations of labeled Rubisco and Rca were utilized in a FRET-based binding assay. Although initial data looked promising, this approach was not fruitful, as no true FRET signal was observed. One possibility is that under the conditions tested, Rca is not able to undergo the structural reorganizations necessary to achieve binding-competent conformations. Rca may also be asymmetric, leading to less stable binding of an already weak interaction.
To better understand the structural adjustments of Rca, subunit exchange between different oligomeric species was examined. It was discovered that subunit exchange is nucleotide dependent, with ADP giving the fastest exchange, ATP giving slower exchange and ATPS inhibiting exchange. Manganese, like ADP, destabilizes subunit-subunit interactions for rapid and facile exchange between oligomers. Three different types of assemblies were deduced from the rates of subunit exchange: rigid types with extremely slow dissociation of individual protomers, tight assemblies with the physiological substrate ATP, and loose assemblies that provide fast exchange due to high ADP.
Information gained about Rca subunit exchange can be used to reexamine the physical interaction between Rubisco and Rca using the FRET-binding assay. These binding assays will provide insight into Rca states able to interact with Rubisco, as well as define conditions to generate bound states for structural analysis. In combination with assembly assays, subunit exchange assays and reactivation studies will provide critical information about the structure/function relationship of Rca in the presence of different nucleotides. Together, these FRET-based assays will help to characterize the Rca regulation mechanism and provide valuable insight into the Rubisco reactivation mechanism.
Preparations of labeled Rubisco and Rca were utilized in a FRET-based binding assay. Although initial data looked promising, this approach was not fruitful, as no true FRET signal was observed. One possibility is that under the conditions tested, Rca is not able to undergo the structural reorganizations necessary to achieve binding-competent conformations. Rca may also be asymmetric, leading to less stable binding of an already weak interaction.
To better understand the structural adjustments of Rca, subunit exchange between different oligomeric species was examined. It was discovered that subunit exchange is nucleotide dependent, with ADP giving the fastest exchange, ATP giving slower exchange and ATPS inhibiting exchange. Manganese, like ADP, destabilizes subunit-subunit interactions for rapid and facile exchange between oligomers. Three different types of assemblies were deduced from the rates of subunit exchange: rigid types with extremely slow dissociation of individual protomers, tight assemblies with the physiological substrate ATP, and loose assemblies that provide fast exchange due to high ADP.
Information gained about Rca subunit exchange can be used to reexamine the physical interaction between Rubisco and Rca using the FRET-binding assay. These binding assays will provide insight into Rca states able to interact with Rubisco, as well as define conditions to generate bound states for structural analysis. In combination with assembly assays, subunit exchange assays and reactivation studies will provide critical information about the structure/function relationship of Rca in the presence of different nucleotides. Together, these FRET-based assays will help to characterize the Rca regulation mechanism and provide valuable insight into the Rubisco reactivation mechanism.
ContributorsForbrook, Dayna S (Author) / Wachter, Rebekka M. (Thesis advisor) / Allen, James (Committee member) / Wang, Xu (Committee member) / Arizona State University (Publisher)
Created2017