CO2 Fixation by Rubisco: Computational Dissection of the Key Steps of Carboxylation, Hydration, and C-C Bond Cleavage
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- 作者：Harald Mauser ; William A. King ; Jill E. Gready ; and T. John Andrews
- 刊名：Journal of the American Chemical Society
- 出版年：2001
- 出版时间：November 7, 2001
- 年：2001
- 卷：123
- 期：44
- 页码：10821 - 10829
- 全文大小：284K
- 年卷期：v.123,no.44(November 7, 2001)
- ISSN：1520-5126
文摘
Despite intensive experimental and computational studies, some important features of the mechanismof the photosynthetic CO2-fixing enzyme, Rubisco, are still not understood. To complement our previousinvestigation of the first catalytic step, the enolization of D-ribulose-1,5-bisphosphate (King et al., Biochemistry1998, 44, 15414-15422), we present the first complete computational dissection of subsequent steps of thecarboxylation reaction that includes the roles of the central magnesium ion and modeled residues of the activesite. We investigated carboxylation, hydration, and C-C bond cleavage using the density functional methodand the B3LYP/6-31G(d) level to perform geometry optimizations. The energies were determined by B3LYP/6-311+G(2d,p) single-point calculations. We modeled a fragment of the active site and substrate, taking intoaccount experimental findings that the residues coordinated to the Mg ion, especially the carbamylated Lys-201, play critical roles in this reaction sequence. The carbamate appears to act as a general base, not only forenolization but also for hydration of the 
ketoacid formed by addition of CO2 and, as well, cleavage of theC2-C3 bond of the hydrate. We show that CO2 is added directly, without assistance of a Michaelis complex,and that hydration of the resultant ketoacid occurs in a separate subsequent step with a discrete transitionstate. We suggest that two conformations of the hydrate (gem-diol), with different metal coordination, arepossible. The step with the highest activation energy during the carboxylation cycle is the C-C bond cleavage.Depending on the conformations of the gem-diol, different pathways are possible for this step. In either case,special arrangements of the metal coordination result in bond breaking occurring at remarkably low activationenergies (between 28 and 37 kcal mol-1) which might be reduced further in the enzyme environment.
ketoacid formed by addition of CO2 and, as well, cleavage of theC2-C3 bond of the hydrate. We show that CO2 is added directly, without assistance of a Michaelis complex,and that hydration of the resultant ketoacid occurs in a separate subsequent step with a discrete transitionstate. We suggest that two conformations of the hydrate (gem-diol), with different metal coordination, arepossible. The step with the highest activation energy during the carboxylation cycle is the C-C bond cleavage.Depending on the conformations of the gem-diol, different pathways are possible for this step. In either case,special arrangements of the metal coordination result in bond breaking occurring at remarkably low activationenergies (between 28 and 37 kcal mol-1) which might be reduced further in the enzyme environment.