科研之友
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摘要
D-Ribulose-1,5-bisphosphate carboxylase/oxygenase (Rubisco) is the most abundant enzyme on Earth and is responsible for the fixation of atmospheric CO2 into biomass. The reaction consists of incorporation of CO2 and solvent H2O into D-ribulose 1,5-bisphosphate (RuBP) to yield 3-phospho-D-glycerate. The reaction involves several proton-dependent events: abstraction and protonation during enolization of RuBP and hydrolysis and reprotonation of the six-carbon reaction intermediate (carboxyketone). Although much is known about Rubisco structure and diversity, fundamental aspects of the reaction mechanism are poorly documented. How and when are protons exchanged among substrate, amino acid residues, and solvent water, and could alterations of proton exchange influence catalytic turnover? What is the energy profile of the reaction? To answer these questions, we measured catalytic rates and the (CO2)-C-12/(CO2)-C-13 isotope effect in isotopic waters. We show that with increasing D2O content, the maximal carboxylation velocity (k(cat)(c)) decreased linearly and was 1.7 times lower in pure D2O. By contrast, the isotope effect on the apparent Michaelis constant for CO2 (K-c) was unity, suggesting that HID exchange might have occurred with the solvent in early steps thereby slowing the overall catalysis. Calculations of kinetic commitments from observed isotope effects further indicate that (1) enolization and processing of the carboxyketone are similarly rate-limiting and (2) the tendency of the carboxyketone to go backward (decarboxylation) is likely exacerbated upon deuteration. Our results thus suggest that Rubisco catalysis is achieved by a rather equal distribution of energy barriers along the reaction.
- 出版日期2013-2-5
- 单位中国地震局
