Rubisco mutants, characterization

In this chapter, we present three approaches to address mechanistic issues with Rubisco mutants characterization of catalysis of partial reactions, analysis of side products, and subtle alteration of the active-site microenvironment by manipulation with exogenous reagents. 

Collectively, these studies illustrate the power of multiple approaches for the analysis of Rubisco mutants. Characterizations of position-329 mutants have not only localized the step of catalysis facilitated by Lys329 but also uncovered its role in intermediate sti)ilization and optimization of carboxylation selectivity. 

Another potential side reaction of the enediol(ate) intermediate is formation of the dicarbonyl compound, l-deoxy-D-glycero-2,3-pentodiulose 5-phosphate, resulting from p-elimination of the Cl-phosphate due to improper stabilization and/or premature dissociation of enediol(ate) from the enzyme active site. This compound has been characterized by reduction with borohydride, oxidation with H2O2, complexation with o-phenylenediamine, and 13C-NMR (23, 34). The p-elimination product is not detected in reactions with wild-type R. rubrum Rubisco but is formed in substantial amounts with mutants in which the Cl-phosphate ligands are substituted, demonstrating the required role of these amino acid side chains in stabilizing the enediol(ate) intermediate (34-35). 

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