Ionization constants of two active-site lysyl epsilon-amino groups of ribulosebisphosphate carboxylase/oxygenase.

Hartman, Fred C.; Milanez, Sylvia; Lee, E H

doi:10.1016/s0021-9258(17)38670-2

Cited by 60 publications

(22 citation statements)

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“…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 ).…”

Section: Discussionmentioning

confidence: 54%

“…Largely on the basis of its observed position and p K a reduction, it has been a long held view that LYS175 is the acid that donates the proton to C2 in the upper 3PGA. − ,, In a previous study, we expressed the opinion that on energetic grounds this pathway was not optimal, but rather, some preliminary model calculations suggested that the protonation could take place more efficiently via the Grotthuss mechanism involving a protonation of P1. The present study, which has identified a specific Grotthuss chain (P1–H 2 O–H 2 O–C2, Figure ), strongly supports this thesis.…”

Section: Discussionmentioning

confidence: 99%

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

Cummins

Gready

2020

J. Phys. Chem. B

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Ribulose 1,5-bisphosphate (RuBP) carboxylase-oxygenase (Rubisco) plays a fundamental role in the carbon cycle by fixing the atmospheric CO 2 used in photosynthesis. Rubisco is all the more remarkable because it must catalyze some difficult multistep reaction chemistry involving proton transfers within the one active site. In the present study, we have used Kohn−Sham density functional theory at the B3LYP/6-31G* level with basis set superposition error and dispersion corrections (B3LYP-gCP-D3) to examine the possibility that the proton transfers can take place through molecular wires (including active-site water molecules) via the classical Grotthuss proton-shuttle mechanism. The results support an essential role for water molecules found in the crystal structures of Rubisco complexes as facilitators of proton transport in all the rate-limiting (catalytic) reaction steps through a network of short proton wires within the Rubisco active site. We suggest that completion of the initial product turnover (cycle) requires two excess protons produced in the initial carbamylation that is required for Rubisco activation. By use of proton wires, a large number of reaction steps may be accommodated within a single active site without necessitating the input of excessive conformational strain energy arising from the movement of residue side chains into positions where direct protonation of substrates can occur. The involvement of the identified types of proton wires in the kinetic mechanism is capable of providing a unique explanation for various experimental observations, including deuterium isotope effects and the results of site-directed mutagenesis experiments, and may thus provide a realistic solution to the problem of Rubisco's challenging chemistry.

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Section: Discussionmentioning

confidence: 54%

Section: Discussionmentioning

confidence: 99%

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

Cummins

Gready

2020

J. Phys. Chem. B

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“…Conserved catalytic residues Glu49, Thr54, Asn112, Lys167, Lys169, Lys192, and Lys330 (Glu60, Thr65, Asn123, Lys175, Lys177, Lys201, and Lys334 in spinach RuBisCO) have all been implicated in various steps of RuBisCO’s catalytic mechanism on the basis of previous biochemical studies. ,,− To delineate the role of these residues in RuBP enolization versus CO 2 fixation steps of the catalytic mechanism, we constructed mutant enzymes E49A, E49G, T54A, N112A, N112K, N112Q, N112S, K167A, K167G, K167R, K169A, K192A, K192C, K192R, and K330A. The identities of amino acid substitutions introduced were largely prompted by previous structure–function studies with the analogous R. rubrum form II RuBisCO. ,− None of these R. palustris form II RuBisCO mutant enzymes restored CO 2 -dependent growth of R. capsulatus strain SB I/II – (Table and Figure S1), demonstrating compromised RuBP carboxylase function. Upon expression in R. rubrum strain IR, photoheterotrophic growth on MTA remained poor for mutants N112S, K167A, K167G, K192A, K192C, and K192R, identical to that seen for the empty plasmid control ( P > 0.05; df = 4) (Table ).…”

Section: Resultsmentioning

confidence: 99%

Structural Perturbations of Rhodopseudomonas palustris Form II RuBisCO Mutant Enzymes That Affect CO₂ Fixation

et al. 2019

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The enzyme ribulose 1,5-bisphosphate carboxylase/oxygenase (RuBisCO) and its central role in capturing atmospheric CO 2 via the Calvin−Benson−Bassham (CBB) cycle have been well-studied. Previously, a form II RuBisCO from Rhodopseudomonas palustris, a facultative anaerobic bacterium, was shown to assemble into a hexameric holoenzyme. Unlike previous studies with form II RuBisCO, the R. palustris enzyme could be crystallized in the presence of the transition state analogue 2-carboxyarabinitol 1,5bisphosphate (CABP), greatly facilitating the structure−function studies reported here. Structural analysis of mutant enzymes with substitutions in form II-specific residues (Ile165 and Met331) and other conserved and semiconserved residues near the enzyme's active site identified subtle structural interactions that may account for functional differences between divergent RuBisCO enzymes. In addition, using a distantly related aerobic bacterial host, further selection of a suppressor mutant enzyme that overcomes negative enzymatic functions was accomplished. Structure−function analyses with negative and suppressor mutant RuBisCOs highlighted the importance of interactions involving different parts of the enzyme's quaternary structure that influenced partial reactions that constitute RuBisCO's carboxylation mechanism. In particular, structural perturbations in an intersubunit interface appear to affect CO 2 addition but not the previous step in the enzymatic mechanism, i.e., the enolization of substrate ribulose 1,5-bisphosphate (RuBP). This was further substantiated by the ability of a subset of carboxylation negative mutants to support a previously described sulfur-salvage function, one that appears to rely solely on the enzyme's ability to catalyze the enolization of a substrate analogous to RuBP.

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“…The original assignment of K334 to the active site and deduction of catalytic functionality were based on its affinity labeling and its displaying of enhanced acidity and nucleophilicity (Norton et al, 1975;Hartman et al, 1985Hartman et al, , 1986. Subsequently, these conclusions were reinforced and extended by site-directed mutagenesis; mutants of the Rhodospirillum rubrum enzyme with replacements for K3342 are unable to carboxylate or oxygenate ribulose-P2 at detectable rates but nevertheless catalyze the "wash out" of tritium from [3-3H]ribulose-P2 at 2-5% of the wild-type rate (Soper et al, 1988;Hartman & Lee, 1989).…”

Section: Y Ho'-t'hmentioning

confidence: 99%

A role for the .epsilon.-amino group of lysine-334 of ribulose-1,5-bisphosphate carboxylase in the addition of carbon dioxide to the 2,3-enediol(ate) of ribulose 1,5-bisphosphonate

Lorimer¹,

Chen²,

Hartman³

1993

Biochemistry

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Earlier structural and functional studies of ribulose-1,5-bisphosphate carboxylase/oxygenase imply that K334 facilitates the addition of gaseous substrate to the 2,3-enediol(ate) derived from ribulose 1,5-bisphosphate. Crystallographic analysis of the activated spinach enzyme [Knight et al. (1990) J. Mol. Biol. 215, 113-160] shows that the lysyl side chain is appropriately positioned to stabilize the transition state for the addition of CO2 to the enediol(ate). Furthermore, despite total impairment of carboxylase and oxygenase activities, site-directed mutants of the Rhodospirillum rubrum enzyme with replacements for lysine K334 (formerly designated K329) retain the capacity to enolize ribulose bisphosphate, demonstrating that the primary catalytic lesion lies beyond this initial step [Soper et al. (1988) Protein Eng. 2, 39-44; Hartman & Lee (1989) J. Biol. Chem. 264, 11784-11789]. We now show that the K334C mutant is also competent in the latter stages of catalysis, whereby 2'-carboxy-3-keto-D-arabinitol 1,5-bisphosphate, the six-carbon intermediate of the carboxylation pathway, is correctly processed to 3-phosphoglycerate. Thus, the impairment of the mutant in overall catalysis can be attributed to preferential disruption of the reaction of CO2 or O2 with the enzyme-bound enediol(ate). Chemical rescue of the K334C mutant by aminoethylation and aminopropylation shows that this disruption reflects, at least in part, a failure to adequately stabilize the relevant transition state. With several simplifying assumptions, the CO2/O2 specificity factor tau can be reduced to the ratio of the fundamental second-order rate constants for the interaction of the gaseous substrates with the enzyme-bound 2,3-enediol(ate) of ribulose bisphosphate.(ABSTRACT TRUNCATED AT 250 WORDS)

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Ionization constants of two active-site lysyl epsilon-amino groups of ribulosebisphosphate carboxylase/oxygenase.

Cited by 60 publications

References 35 publications

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

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

Structural Perturbations of Rhodopseudomonas palustris Form II RuBisCO Mutant Enzymes That Affect CO₂ Fixation

A role for the .epsilon.-amino group of lysine-334 of ribulose-1,5-bisphosphate carboxylase in the addition of carbon dioxide to the 2,3-enediol(ate) of ribulose 1,5-bisphosphonate

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Ionization constants of two active-site lysyl epsilon-amino groups of ribulosebisphosphate carboxylase/oxygenase.

Cited by 60 publications

References 35 publications

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

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

Structural Perturbations of Rhodopseudomonas palustris Form II RuBisCO Mutant Enzymes That Affect CO2 Fixation

A role for the .epsilon.-amino group of lysine-334 of ribulose-1,5-bisphosphate carboxylase in the addition of carbon dioxide to the 2,3-enediol(ate) of ribulose 1,5-bisphosphonate

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Structural Perturbations of Rhodopseudomonas palustris Form II RuBisCO Mutant Enzymes That Affect CO₂ Fixation