Multiple catalytic roles of His 287 of Rhodospirillum rubrum ribulose 1,5-bisphosphate carboxylase/oxygenase.

Active-site His 287 of Rhodospirillum rubrum ribulose 1,5-bisphosphate (RuBP) carboxylase/oxygenase interacts with the C3-hydroxyl of bound substrate or reaction-intermediate analogue (CABP), water molecules, and ligands for the activator metal-ion (Andersson I, 1996, J Mol Biol 259:160-174; Taylor TC, Andersson I, 1997, J Mol Biol 265:432-444). To test structure-based postulates of catalytic functionality, His 287 was replaced with Asn or Gln. The mutants are not affected adversely in subunit assembly, activation (binding of Mg2+ and carbamylation of Lys 191), or recognition of phosphorylated ligands; they bind CABP with even greater tenacity than does wild-type enzyme. H287N and H287Q are severely impaired in catalyzing overall carboxylation (approximately 10(3)-fold and > 10(5)-fold, respectively) and enolization (each mutant below threshold for detection) of RuBP. H287N preferentially catalyzes decarboxylation of carboxylated reaction intermediate instead of forward processing to phosphoglycerate. Analysis of RuBP turnover that occurs at high concentrations of mutants over extended time periods reveal > 10-fold reduced CO2/O2 specificities, elevated misprotonation of the enediol intermediate, and misprocessing of the oxygenated intermediate of the oxygenase pathway. These results are consistent with multifaceted roles for His 287 in promoting enediol formation, enediol tautomerization, and forward-processing of carboxylated intermediate.

A reconstruction of the gene for ribulose bisphosphate carboxylase from Rhodospirillum rubrum that expresses the authentic enzyme in Escherichia coli.

Escherichia coli plasmid pRR36, which expresses Rhodospirillum rubrum ribulose bisphosphate carboxylase/oxygenase (EC 4.1.1.39) as a fusion protein [Nargang et al., Mol. Gen. Genet. 193 (1984) 220-224], was used to construct a new clone of the carboxylase gene (rbc) whose expression product is the wild-type enzyme. This construction entailed removing all lacZ-coding sequences and a portion of the 5'-noncoding leader of the R. rubrum rbc gene. The highest specific activity of carboxylase was observed with an expression vector which juxtaposed the trp-lac (tac) hybrid promoter with the R. rubrum ribosome binding site and the rbc structural gene. The carboxylase expressed in E. coli JM107 was purified to near homogeneity and, based on subunit Mr and specific enzymic activity, the isolated protein appeared indistinguishable from authentic ribulose bisphosphate carboxylase from R. rubrum. N-terminal sequence analyses of the cloned enzyme verified that the cloned and wild-type enzymes are the same.

Interaction of isocitrate dehydrogenase with (RS)-3-bromo-2-ketoglutarate. A potential affinity label for alpha-ketoglutarate binding sites.

The interaction of oxidized nicotine adenine dinucleotide phosphate dependent isocitrate dehydrogenase (from pig heart) with (RS)-3-bromo-2-ketoglutarate was investigated in an effort to evaluate the reagent's potential as a selective reagent for alpha-ketoglutarate binding sites. The enzyme is rapidly inactivated by 0.1 mM bromoketoglutarate at pH 7.4. With increasing concentrations of regent, the reaction shows a rate saturation; the minimum inactivation half-time is 3 min and Kinact for bromoketoglutarate is 250 microM. Isocitrate and NADP+ protect against inactivation, while ketoglutarate does not. When tested in the assay that monitors isocitrate oxidation, bromoketoglutarate is a competitive inhibitor (Ki = 100 microM) of the dehydrogenase. As judged by oxidation of NADPH, bromoketoglutarate is also a substrate for isocitrate dehydrogenase, exhibiting a Km of 250 microM and a Vmax comparable to that for isocitrate oxidation. The reduction of bromoketoglutarate is competitively inhibited by isocitrate (Ki = 3 microM) and ketoglutarate (Ki = 50 microM). Like the enzyme-catalyzed oxidation of isocitrate, the reduction of bromoketoglutarate is stereospecific, requires divalent metal ions, and shows absolute specificity for NADPH. However, since CO2 is not required for catalytic turnover of bromoketoglutarate, its reduction is likely comparable to that of oxalosuccinate rather than the reductive carboxylation of ketoglutarate. Although bromoketoglutarate, as a substrate for isocitrate dehydrogenase, clearly has affinity for the active site, the irreversible inactivation of the enzyme by the reagent may result from modification outside the active-site region, since inactivation during catalytic turnover of bromoketoglutarate is not observed. Commercial isocitrate dehydrogenase is purified 12-fold by affinity chromatography on thiol-agarose alkylated by bromoketoglutarate.

Demonstration of a functional requirement for the carbamate nitrogen of ribulosebisphosphate carboxylase/oxygenase by chemical rescue.

Ribulosebisphosphate carboxylase/oxygenase is reversibly activated by the reaction of CO2 with a specific lysyl residue (Lys191 of the Rhodospirillum rubrum enzyme) to form a carbamate that coordinates an essential Mg2+ cation. Surprisingly, the Lys191----Cys mutant protein, in the presence of CO2 and Mg2+, exhibits tight binding of the reaction intermediate analogue 2-carboxyarabinitol bisphosphate [Smith, H. B., Larimer, F. W., & Hartman, F. C. (1988) Biochem. Biophys. Res. Commun. 152, 579-584], a property normally equated with effective coordination of the Mg2+ by the carbamate. Catalytic ineptness of the Cys191 mutant protein, despite its ability to coordinate Mg2+ properly, might be due to the absence of the carbamate nitrogen. To investigate this possibility, we have evaluated the ability of exogenous amines to restore catalytic activity to the mutant protein. Significantly, the Cys191 protein manifests ribulose bisphosphate dependent fixation of 14CO2 when incubated with aminomethanesulfonate but not ethanesulfonate. This novel activity reflects a Km value for ribulose bisphosphate which is not markedly perturbed relative to wild-type enzyme, a Km for Mg2+ which is in fact decreased 10-fold, and rate saturation with respect to aminomethanesulfonate (Kd = 8 mM). Chromatographic and spectrophotometric analyses reveal the product of CO2 fixation to be D-3-phosphoglycerate, while turnover of [1-3H]ribulose bisphosphate into [3H]phosphoglycolate confirms oxygenase activity. We conclude that aminomethanesulfonate restored ribulosebisphosphate carboxylase/oxygenase activities to the Cys191 mutant protein by providing a nitrogenous function which satisfies a catalytic demand normally met by the carbamate nitrogen of Lys191.

Catalytic nonessentiality of an active-site cysteinyl residue of phosphoribulokinase.

The activity of the Calvin cycle enzyme phosphoribulokinase is coupled to photosynthetic electron transport by reversible oxidation/reduction mediated by thioredoxin-f. Previous studies have shown that one of the regulatory sulfhydryl groups, that of Cys-16, is positioned at the nucleotide-binding domain of the active site. To determine if oxidative deactivation of the kinase reflects catalytic essentiality of Cys-16, the methylation of spinach phosphoribulokinase by methyl-4-nitrobenzenesulfonate has been examined. Methylation of the kinase results in a 50% loss of the initial activity relative to controls. The suppression of kcat is accompanied by a 6-fold increase in the Km for ATP, without change in the Km for ribulose 5-phosphate. The insensitivity of the modified enzyme, in contrast to the native, to iodoacetate and 5,5'-dithiobis(2-nitrobenzoate) indicates that Cys-16 is a site of methylation. This supposition is verified independently by peptide mapping and Edman degradation subsequent to S-carboxymethylation with [14C]iodoacetate of the methylated kinase. Retention of significant enzymatic activity after complete modification of Cys-16 with the small, uncharged methyl moiety demonstrates that this active-site residue is not essential for catalysis.

Evidence supporting lysine 166 of Rhodospirillum rubrum ribulosebisphosphate carboxylase as the essential base which initiates catalysis.

The epsilon-amino group of Lys-166 of Rhodospirillum rubrum ribulosebisphosphate carboxylase/oxygenase was postulated as the essential base which initiates catalysis by abstracting the proton at C-3 of ribulose 1,5-bisphosphate (Hartman, F. C., Soper, T. S., Niyogi, S. K., Mural, R. J., Foote, R. S., Mitra, S., Lee, E. H., Machanoff, R., and Larimer, F. W. (1987) J. Biol. Chem. 262, 3496-3501). To scrutinize this possibility, the site-directed Gly-166 mutant, totally devoid of ribulosebisphosphate carboxylase activity, was examined for its ability to catalyze each of three partial reactions. When carbamylated at Lys-191 (i.e. activated with CO2 and Mg2+), wild-type enzyme catalyzed the hydrolysis of 2-carboxy-3-keto-D-arabinitol 1,5-bisphosphate, the six-carbon reaction intermediate of the carboxylase reaction (Pierce, J., Andrews, T. J., and Lorimer, G. H. (1986a) J. Biol. Chem. 261, 10248-10256). Likewise, when carbamylated at Lys-191, the Gly-166 mutant also catalyzed the hydrolysis of this reaction intermediate. The carbamylated wild type catalyzed the enolization of ribulose 1,5-bisphosphate as indicated by the transfer of 3H radioactivity from [3-3H]ribulose, 1,5-bisphosphate to the medium. However, even when carbamylated at Lys-191, the mutant protein did not catalyze the enolization of ribulose 1,5-bisphosphate. Additionally, unlike the decarbamylated wild-type enzyme, which catalyzed the decarboxylation of 2-carboxy-3-keto-D-arabinitol 1,5-bisphosphate in the absence of Mg2+, the mutant protein was inactive in this partial reaction. These properties exclude the epsilon-amino group of Lys-166 as an obligatory participant in the hydrolysis of 2-carboxy-3-keto-D-arabinitol 1,5-bisphosphate. In contrast, these properties are consistent with the epsilon-amino group of Lys-166 functioning as an acid-base catalyst in the enolization of ribulose 1,5-bisphosphate (when the enzyme is carbamylated) and in the decarboxylation of 2-carboxy-3-keto-D-arabinitol 1,5-bisphosphate (when the enzyme is decarbamylated). Alternatively, Lys-166 may stabilize the transition states of these two partial reactions.

Enhanced CO2/O2 specificity of a site-directed mutant of ribulose-bisphosphate carboxylase/oxygenase.

The CO2/O2 specificity factor of ribulose-bisphosphate carboxylase/oxygenase partially determines the efficiency of photosynthetic carbon assimilation. Heretofore, engineered alterations of the enzyme have only decreased the selectivity for CO2 utilization. We show that alanyl replacement of active-site Ser-368 of the Rhodospirillum rubrum carboxylase enhances the carboxylation selectivity approximately 1.6-fold over the wild-type level. This enhancement reflects a greater relative decline in oxygenase efficiency than in carboxylase efficiency. In contrast to wild-type enzyme, the carboxylase activity of the Ser-368 mutant protein is not perceptibly inhibited by O2, perhaps indicative of a change in rate-limiting steps in the overall reaction pathway.

Examination of the function of active site lysine 329 of ribulose-bisphosphate carboxylase/oxygenase as revealed by the proton exchange reaction.

Diverse approaches that include site-directed mutagenesis have indicated a catalytic role of Lys-329 of ribulosebisphosphate carboxylase/oxygenase from Rhodospirillum rubrum. To determine whether Lys-329 is required for the initial enolization of ribulose bisphosphate or for some subsequent step in the overall reaction pathway, the competence of position 329 mutant proteins (devoid of carboxylase activity) in catalyzing exchange of solvent protons with the C-3 proton of substrate has now been examined. Irrespective of the amino acid substitution for Lys-329, the mutant protein retains 2-6% of the wild-type activity in the proton exchange reaction. The complete stability of ribulose bisphosphate during the enolization catalyzed by mutant protein suggests that the major effect of Lys-329 is to facilitate the addition of gaseous substrates (CO2 or O2) to the enediol intermediate. The exchange reaction requires Mg2+, is CO2-dependent, and is inhibited by the transition-state analogue 2-carboxyarabinitol 1,5-bisphosphate. A mutant protein in which Lys-191, the site for carbamylation by CO2 in an obligatory activation step, is replaced by a cysteinyl residue totally lacks proton exchange activity. Barely detectable exchange activity (approximately 0.2% of wild-type) is displayed by the Lys-166----Cys mutant protein, consistent with the previously implicated role of Lys-166 in the deprotonation of ribulose bisphosphate. Retention of exchange activity by the Glu-48----Gln mutant protein, which is slightly active in overall carboxylation, demonstrates that active site Glu-48, like Lys-329, exerts its major effect at some step subsequent to the initial enolization.

A signature of the oxygenase intermediate in catalysis by ribulose-bisphosphate carboxylase/oxygenase as provided by a site-directed mutant.

An uncharacterized minor transient product, observed in our earlier studies of substrate turnover by the E48Q mutant of Rhodospirillum rubrum ribulose-bisphosphate carboxylase/oxygenase (Lee, E. H., Harpel, M. R., Chen, Y.-R., and Hartman, F. C. (1993) J. Biol. Chem. 268, 26583-26591), becomes a major product when it is trapped and stabilized with borate as an additive to the reaction mixture. Chemical characterization establishes this novel product as D-glycero-2,3-pentodiulose 1,5-bisphosphate, thereby demonstrating oxidation of the C-3 hydroxyl of D-ribulose 1,5-bisphosphate to a carbonyl. As the formation of the novel oxidation product is oxygen-dependent and generates hydrogen peroxide, its precursor must be a peroxy derivative of ribulose bisphosphate. Thus, discovery of the dicarbonyl bisphosphate lends direct support to the long standing, but heretofore unproven, postulate that the normal pathway for oxidative cleavage of ribulose bisphosphate by the wild-type enzyme entails a peroxy intermediate. Our results also suggest that stabilization of the peroxy intermediate by the wild-type enzyme promotes carbon-carbon scission as opposed to elimination of hydrogen peroxide.

Restoration of activity to catalytically deficient mutants of ribulosebisphosphate carboxylase/oxygenase by aminoethylation.

Substitutions for active-site lysyl residues at positions 166 and 329 in ribulosebisphosphate carboxylase/oxygenase from Rhodospirillum rubrum have been shown to abolish catalytic activity. Treatment of the Cys-166 and Cys-329 mutant proteins with 2-bromoethylamine partially restores enzyme activity, presumably as a consequence of selective aminoethylation of the thiol group unique to each protein. Amino acid analyses, slow inactivation of the wild-type carboxylase by bromoethylamine, and the failure of bromoethylamine to restore activity to the corresponding glycyl mutant proteins support this interpretation. The observed facile, selective aminoethylations may reflect an active site microenvironment not dissimilar to that of the native enzyme. Catalytic constants of these novel carboxylases, which contain a sulfur atom in place of a specific lysyl gamma-methylene group, are significantly lower than that of the wild-type enzyme. Furthermore, the aminoethylated mutant proteins form isolable complexes with a transition state analogue, but with compromised stabilities. These detrimental effects by such a modest structural change underscore the stringent requirement for lysyl side chains at positions 166 and 329. In contrast, the aminoethylated mutant proteins exhibit carboxylase/oxygenase activity ratios and Km values that are unperturbed relative to those for the native enzyme.

Facilitation of the terminal proton transfer reaction of ribulose 1,5-bisphosphate carboxylase/oxygenase by active-site Lys166.

The terminal step in the carboxylation pathway catalyzed by ribulose 1,5-bisphosphate carboxylase/oxygenase (Rubisco) is stereospecific protonation of the C-2 aci-acid of 3-phosphoglycerate (PGA). X-ray crystallographic results favor the epsilon-amino group of Lys166 as the proton donor in this step [Knight et al. (1990) J. Mol. Biol. 215, 113]. Nonetheless, position-166 mutants are able to catalyze forward processing of isolated 2-carboxy-3-ketoarabinitol 1,5-bisphosphate (CKABP), the carboxylated reaction intermediate [Lorimer G.H., & Hartman, F.C. (1988) J. Biol. Chem. 263, 6468]. Prior assays for intermediate processing relied solely on formation of acid-stable radioactivity from acid-labile [2'-14C]CKABP. Therefore, PGA, the normal reaction product, may not have been distinguished from pyruvate, the product from beta-elimination of phosphate from the terminal aci-acid intermediate [Andrews, T.J., & Kane, H.J. (1991) J. Biol. Chem. 266, 9447]. If Lys166 indeed serves as the terminal proton donor, mutants lacking an ionizable side chain at position 166 might process the carboxylated intermediate predominantly to pyruvate. We have thus used anion exchange chromatography and enzyme coupling to separate and identify the products from turnover of [2'-14C]CKABP by wild-type, K166G, and K166S enzymes. Although PGA is the only labeled product of significance formed by wild-type enzyme, pyruvate is a major labeled product formed by the mutants. These results provide the first direct functionally-based evidence that Lys166 is crucial to the last step in Rubisco-catalyzed conversion of RuBP to PGA.

Isolation and sequencing of an active-site peptide from Rhodospirillum rubrum ribulosebisphosphate carboxylase/oxygenase after affinity labeling with 2-[(bromoacetyl)amino]pentitol 1,5-bisphosphate.

2-[(Bromoacetyl)amino]pentitol 1,5-bisphosphate was reported to be a highly selective affinity label for ribulosebisphosphate carboxylase/oxygenase from Rhodospirillum rubrum [Fraij, B., & Hartman, F. C. (1982) J. Biol. Chem. 257, 3501-3505]. The enzyme has now been inactivated with a 14C-labeled reagent in order to identify the target residue at the sequence level. Subsequent to inactivation, the enzyme was carboxymethylated with iodoacetate and then digested with trypsin. The only radioactive peptide in the digest was obtained at a high degree of purity by successive chromatography on DEAE-cellulose, SP-Sephadex, and Sephadex G-25. On the basis of amino acid analysis of the purified peptide, the derivatized residue was a methionyl sulfonium salt. Automated Edman degradation confirmed the purity of the labeled peptide and established its sequence as Leu-Gln- Gly-Ala-Ser-Gly-Ile-His-Thr-Gly-Thr-Met-Gly-Phe-Gly-Lys-Met-Glu-Gly-Glu-Ser-Ser - Asp-Arg. Cleavage of this peptide with cyanogen bromide showed that the reagent moiety was covalently attached to the second methionyl residue. Sequence homology with the carboxylase/oxygenase from spinach indicates that the lysyl residue immediately preceding the alkylated methionine corresponds to Lys-334, a residue previously implicated at the active site.

2-(4-Bromoacetamido)anilino-2-deoxypentitol 1,5-bisphosphate, a new affinity label for ribulose bisphosphate carboxylase/oxygenase from Rhodospirillum rubrum. Determination of reaction parameters and characterization of an active site peptide.

A new affinity label for ribulose bisphosphate carboxylase/oxygenase from Rhodospirillum rubrum, 2-(4-bromoacetamido)anilino-2-deoxypentitol 1,5-bisphosphate, has been prepared, Reductive amination of ribulose-P2 with p-phenylenediamine in the presence of sodium cyanoborohydride yielded an epimeric mixture which was resolved by chromatography on quaternary aminoethyl-Sephadex. Subsequent bromoacetylation of the isolated amino bisphosphates gave reagents A and B (ribo and arabino epimers of 2-(4-bromoacetamido) anilino-2-deoxypentitol 1,5-bisphosphate) which were competitive inhibitors of the carboxylase with Ki values of 705 and 104 microM, respectively. Reagent A exhibited no time-dependent effects on the carboxylase in either the deactivated or activated state. Incubation of the enzyme with reagent B in the presence of the essential activators CO2 and Mg2+, however, resulted in an irreversible, time-dependent loss of activity, with a Kinact of 125 microM and a minimal half-time of 7.3 min. Covalent incorporation of [14C]reagent B was directly proportional to the loss of activity, with total inactivation correlating with an incorporation of 1.1 mol of reagent/mol of subunit. Inclusion of the competitive inhibitor 2-carboxyribitol 1,5-bisphosphate protected against inactivation with a concomitant reduction in incorporation. Neither reagent affected the activity of spinach carboxylase. Fractionation of [14C]reagent B-modified enzyme on DEAE-cellulose, subsequent to carboxymethylation and tryptic digestion, revealed two major radioactive peaks of approximately equal area. Digestion of each peak with alkaline phosphatase and rechromatography on DEAE-cellulose resulted in pure peptides I and II. The peptides were identical except in the site of labeling: peptide I contained a modified cysteinyl residue while peptide II contained a modified histidyl residue. Automated Edman degradation established the sequence as (sequence in text) which is located near the NH2 terminus of the enzyme. The lack of reactivity with the spinach enzyme is explained by the deletion of the histidyl residue and the replacement of cysteine by tryptophan in the eukaryotic species. Although the nonconservation of the modified residues argues against a functional role other than maintenance of structural integrity, the extensive homology in this region among seven different species of carboxylase is compatible with the region comprising a portion of the active site.