Multifaceted Roles of Lys166 of Ribulose-bisphosphate Carboxylase/Oxygenase As Discerned by Product Analysis and Chemical Rescue of Site-Directed Mutants

Abstract

Ab initio calculations [King, W. A., et al. (1998) Biochemistry 37, 15414-15422] of an active-site mimic of D-ribulose-1,5-bisphosphate carboxylase/oxygenase suggest that active-site Lys166 plays a role in carboxylation in addition to its functions in the initial deprotonation and final protonation steps. To test this postulate, the turnover of 1-(3)H-labeled D-ribulose 1,5-bisphosphate (RuBP) by impaired position-166 mutants was characterized. Although these mutants catalyze slow enolization of RuBP, most of the RuBP-enediol undergoes beta-elimination of phosphate to form 2,3-pentodiulose 5-phosphate, signifying deficiencies in normal carboxylation and oxygenation. Much of the remaining RuBP-enediol is carboxylated but forms pyruvate, rather than 3-phospho-D-glycerate, due to incapacity in protonation of the terminal aci-acid intermediate. As a further test of the postulate, the effects of subtle perturbation of the Lys166 side chain on the carboxylation/oxygenation partitioning ratio (tau) were determined. To eliminate a chemically reactive site, Cys58 was replaced by a seryl residue without any loss of activity. The virtually inactive K166C-C58S double mutant was chemically rescued by aminoethylation or aminopropylation to reinsert a lysyl-like side chain at position 166. Relative to the wild-type value, tau for the aminoethylated enzyme was increased by approximately 30%, and tau for the aminopropylated enzyme was decreased by approximately 80%. Thus, two lines of experimentation support the theoretically based conclusion for the importance of Lys166 in the reaction of RuBP-enediol with gaseous substrates.

Kohn–Sham Density Functional Calculations Reveal Proton Wires in the Enolization and Carboxylase Reactions Catalyzed by Rubisco

“…The present study, which has identified a specific Grotthuss chain (P1–H 2 O–H 2 O–C2, Figure ), strongly supports this thesis. Furthermore, although the typical lysine p K a of approximately 10.5 is lowered to 7.9 in LYS175, site-directed mutagenesis suggests LYS175 may not be absolutely necessary for product formation. , It has also been thought that LYS175 directly protonates O2 in the enolization of RuBP. − ,,, However, the results of the present study demonstrate that, throughout the course of the reaction, LYS175 could well H bond the P1 phosphate rather than O2 (e.g., in Figures and and Supporting Information). Alternatively, enolization can be achieved without difficulty by a single base, KCX201, through a network of proton wires (H3–H 2 O–KCX201 and KCX201–H 2 O–H 2 O–O2, Figure ).…”

Oxygen toxicity from plants to people

“…However, premature formation of the ene-diolate is likely to result in elimination of the C-1 phosphate ester. Rubisco has minimized this potential problem by coupling capture of carbon dioxide with proton transfer to the enzyme's essential base (lysine 166, see Harpel et al 2002). This would also solve another of Rubisco's remaining mysteries: why is the essential base used for proton abstraction placed distant from C-3 of ribulose bisphosphate?…”

Ab Initio Molecular Dynamics Simulation and Energetics of the Ribulose-1,5-biphosphate Carboxylation Reaction Catalyzed by Rubisco: Toward Elucidating the Stereospecific Protonation Mechanism

“…The enzyme ribulose 1,5-bisphosphate carboxylase–oxygenase (Rubisco), as the main route for fixation of atmospheric carbon into biological carbon in plant photosynthesis, is an attractive target for re-engineering for improved efficiency and potentially greater crop productivity. , The successful re-engineering of an enzyme is highly dependent on understanding the catalytic function(s) of Rubisco’s totally conserved active-site residues. One such residue, LYS175 (in spinach Rubisco), has been implicated in various phases of the complex multistep reaction catalyzed by Rubisco . In particular, structural analysis reveals that LYS175 is well positioned for the stereospecific protonation that completes the formation of product; consequently, it is considered to be the most likely, if not the only, amino-acid proton donor available to perform this function. , Our proposed mechanism of Rubisco catalysis is summarized in Figure .…”

“…One such residue, LYS175 (in spinach Rubisco), has been implicated in various phases of the complex multistep reaction catalyzed by Rubisco. 3 In particular, structural analysis reveals that LYS175 is well positioned for the stereospecific protonation that completes the formation of product; consequently, it is considered to be the most likely, if not the only, amino-acid proton donor available to perform this function. 4,5 Our proposed mechanism of Rubisco catalysis is summarized in Figure 1.…”