Kinetic and mutational analyses of the regulation of phosphoribulokinase by thioredoxins.

Despite little supportive data, differential target protein susceptibility to redox regulation by thioredoxin (Trx) f and Trx m has been invoked to account for two distinct Trxs in chloroplasts. However, this postulate has not been rigorously tested with phosphoribulokinase (PRK), a fulcrum for redox regulation of the Calvin cycle. Prerequisite to Trx studies, the activation of spinach PRK by dithiothreitol, 2-mercaptoethanol, and glutathione was examined. Contrary to prior reports, each activated PRK, but only dithiothreitol supported Trx-dependent activation. Comparative kinetics of activation of PRK showed Trx m to be more efficient than Trx f because of its 40% higher V(max) but similar S(0.5). Activations were insensitive to ribulosebisphosphate carboxylase, which may complex with PRK in vivo. To probe the basis for superiority of Trx m, we characterized site-directed mutants of Trx f, in which unique residues in conserved regions were replaced with Trx m counterparts or deleted. These changes generally resulted in V(max) enhancements, the largest (6-fold) of which occurred with T105I, reflective of substitution in a hydrophobic region that opposes the active site. Inclusive of the present study, activation kinetics of several different Trx-regulated enzymes indicate redundancy in the functions of the chloroplastic Trxs.

D-ribose-5-phosphate isomerase from spinach: heterologous overexpression, purification, characterization, and site-directed mutagenesis of the recombinant enzyme.

A cDNA encoding spinach chloroplastic ribose-5-phosphate isomerase (RPI) was cloned and overexpressed in Escherichia coli, and a purification scheme for the recombinant enzyme was developed. The purified recombinant RPI is a homodimer of 25-kDa subunits and shows kinetic properties similar to those of the homodimeric enzyme isolated from spinach leaves (A. C. Rutner, 1970, Biochemistry 9, 178-184). Phosphate, used as a buffer in previous studies, is a competitive inhibitor of RPI with a K(i) of 7.9 mM. D-Arabinose 5-phosphate is an effective inhibitor, while D-xylulose-5 phosphate is not, indicating that the configuration at carbon-3 contributes to substrate recognition. Although D-arabinose 5-phosphate binds to RPI, it is not isomerized, demonstrating that the configuration at carbon-2 is crucial for catalysis. Alignment of RPI sequences from diverse sources showed that only 11 charged amino acid residues of the 236-residue subunit are conserved. The possible function of four of these residues was examined by site-directed mutagenesis. D87A, K100A, and D90A mutants show greatly diminished k(cat) values (0. 0012, 0.074, and 0.38% of the wild type, respectively), while E91A retains substantial activity. Only insignificant or moderate changes in K(m) of D-ribose 5-phosphate are observed for D87A, K100A, and D90A, indicating a direct or indirect catalytic role of the targeted residues.

Oxidation-reduction properties of chloroplast thioredoxins, ferredoxin:thioredoxin reductase, and thioredoxin f-regulated enzymes.

Oxidation-reduction midpoint potentials were determined, as a function of pH, for the disulfide/dithiol couples of spinach and pea thioredoxins f, for spinach and Chlamydomonas reinhardtii thioredoxins m, for spinach ferredoxin:thioredoxin reductase (FTR), and for two enzymes regulated by thioredoxin f, spinach phosphoribulokinase (PRK) and the fructose-1,6-bisphosphatases (FBPase) from pea and spinach. Midpoint oxidation-reduction potential (Em) values at pH 7.0 of -290 mV for both spinach and pea thioredoxin f, -300 mV for both C. reinhardtii and spinach thioredoxin m, -320 mV for spinach FTR, -290 mV for spinach PRK, -315 mV for pea FBPase, and -330 mV for spinach FBPase were obtained. With the exception of spinach FBPase, titrations showed a single two-electron component at all pH values tested. Spinach FBPase exhibited a more complicated behavior, with a single two-electron component being observed at pH values >/= 7.0, but with two components being present at pH values <7.0. The slopes of plots of Em versus pH were close to the -60 mV/pH unit value expected for a process that involves the uptake of two protons per two electrons (i. e., the reduction of a disulfide to two fully protonated thiols) for thioredoxins f and m, for FTR, and for pea FBPase. The slope of the Em versus pH profile for PRK shows three regions, consistent with the presence of pKa values for the two regulatory cysteines in the region between pH 7.5 and 9.0.

Identification of a catalytic aspartyl residue of D-ribulose 5-phosphate 3-epimerase by site-directed mutagenesis.

Guided by comparative sequence considerations, we have examined the possibility of a catalytic role of Asp186 of D-ribulose 5-phosphate epimerase by site-directed mutagenesis of the recombinant spinach enzyme. Accordingly, D186A, D186N, and D186E mutants of the epimerase were constructed, purified, and characterized; as judged by their electrophoretic mobilities the mutants are properly assembled into octamers like the wild-type enzyme. Based on the extent of internal quenching of Trp fluorescence, the conformational integrity of the wild-type enzyme is preserved in the mutants. The wild-type kcat of 7.1 x 10(3) s-1 is lowered to 3.3 x 10(-4) s-1 in D186A, 0.13 s-1 in D186N, and 1.1 s-1 in D186E; as gauged by D186A, altogether lacking a functional side chain at position 186, the beta-carboxyl of Asp186 facilitates catalysis by >10(7)-fold. Relative to the wild-type enzyme, the Km for D-ribulose 5-phosphate is essentially unaltered with D186N and D186E but increased 10-fold with D186A. Apart from their impairments in epimerase activity, the mutants are unable to catalyze exchange between solvent protons and the C3 proton of substrates. This deficiency and the differential alterations of kinetic parameters among the mutants are consistent with Asp186 serving as an electrophile to facilitate alpha-proton abstraction. This study is the first to identify a catalytic group of the epimerase.

D-Ribulose-5-phosphate 3-epimerase: cloning and heterologous expression of the spinach gene, and purification and characterization of the recombinant enzyme.

We have achieved, to our knowledge, the first high-level heterologous expression of the gene encoding D-ribulose-5-phosphate 3-epimerase from any source, thereby permitting isolation and characterization of the epimerase as found in photosynthetic organisms. The extremely labile recombinant spinach (Spinacia oleracea L.) enzyme was stabilized by DL-alpha-glycerophosphate or ethanol and destabilized by D-ribulose-5-phosphate or 2-mercaptoethanol. Despite this lability, the unprecedentedly high specific activity of the purified material indicates that the structural integrity of the enzyme is maintained throughout isolation. Ethylenediaminetetraacetate and divalent metal cations did not affect epimerase activity, thereby excluding a requirement for the latter in catalysis. As deduced from the sequence of the cloned spinach gene and the electrophoretic mobility under denaturing conditions of the purified recombinant enzyme, its 25-kD subunit size was about the same as that of the corresponding epimerases of yeast and mammals. However, in contrast to these other species, the recombinant spinach enzyme was octameric rather than dimeric, as assessed by gel filtration and polyacrylamide gel electrophoresis under nondenaturing conditions. Western-blot analyses with antibodies to the purified recombinant enzyme confirmed that the epimerase extracted from spinach leaves is also octameric.

Roles and microenvironments of tryptophanyl residues of spinach phosphoribulokinase.

Phosphoribulokinase is one of several Calvin cycle enzymes that are light-regulated via the ferredoxin-thioredoxin system (R. A. Wolosiuk and B. B. Buchanan, 1978, Arch. Biochem. Biophys. 189, 97-101). Substitution of the only two Trp residues of the enzyme was prompted by the following goals: to identify each tryptophanyl residue with respect to prior classifications as exposed and buried (C. A. Ghiron et al., 1988, Arch. Biochem. Biophys. 260, 267-272); to explore the possible active-site location and function of conserved Trp155, as suggested by sequence proximity to catalytic Asp160 (H. A. Charlier et al., 1994, Biochemistry 33, 9343-9350); and to determine if fluorescence of a Trp residue can serve as a gauge of conformational differences between the reduced (active) and the oxidized (inactive) forms of the enzyme. Emission spectra and acrylamide quenching data demonstrate that Trp155 is solvent exposed, while Trp241 is buried. Kinetic parameters of the W241F mutant are not significantly altered relative to those of wild-type enzyme, thereby discounting any requirement for Trp at position 241. While substitution of Trp155 with Phe or Ala has little impact on Vmax, the Km for Ru5P and ATP are increased substantially; the diminished affinity for ATP is particularly pronounced in the case of the Ala substitution. In further support of an active-site location of Trp155, its fluorescence emission is subject to quenching by nucleotides. Fluorescence quenching of reduced W241F by ATP gives a dissociation constant (Kd) of 37 microM, virtually identical with its Km of 46 microM, and provides for the first time a direct measurement of the interaction of the kinase with product ADP (Kd of 1.3 mM). Fluorescence quenching of oxidized W241F by ATP reveals a Kd of 28 mM; however, this weakened binding does not reflect an altered microenvironment of Trp155, as its steady-state emission and fluorescence lifetimes are unaffected by the oxidation state.

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.

Oxidation-reduction properties of the regulatory site of spinach phosphoribulokinase.

The oxidation-reduction midpoint potential (Em) of the regulatory disulfide, formed between Cys16 and Cys55, of spinach chloroplast phosphoribulokinase has been determined both for the wild-type enzyme and for a C244S-C250S double mutant, using enzymatic activity to monitor the oxidation-reduction state of the regulatory disulfide. At pH 7.0, Em values for the two-electron reduction of the regulatory disulfide of -295 +/- 10 and -290 +/- 10 mV were measured for the wild-type and mutant, respectively. In contrast to the dependence of activity on ambient potential (Eh) observed for the wild-type enzyme and the double mutant, which both followed the Nernst equation for a two-electron process, high and constant activity was exhibited by a C16S-C244S-C250 triple mutant of the enzyme at all Eh values tested. Em values for the wild-type enzyme were also measured at pH values of 6.7, 7.5, 7.7, and 8.2 and the Em vs pH data in this region give a good fit to a straight line with a slope of -60 mV/pH unit.

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.

Identification of residues of spinach thioredoxin f that influence interactions with target enzymes.

The necessity for two types of thioredoxins (Trx f and m) within chloroplasts of higher plants that mediate the same redox chemistry with various target enzymes is not well understood. To approach this complex issue, we have applied site-directed mutagenesis to the identification of residues of Trx f that affect its binding to and selectivity for target enzymes. Based upon amino acid sequence alignments and the three-dimensional structure of Escherichia coli thioredoxin, putative key residues of Trx f were replaced with residues found at corresponding positions of Trx m to generate the mutants K58E, Q75D, N74D, and deletion mutants DeltaAsn-74 and DeltaAsn-77. Kinetics of activation of oxidized recombinant sorghum leaf NADP-dependent malate dehydrogenase and oxidized spinach chloroplastic fructose-1,6-bisphosphatase by wild-type Trx f, wild-type Trx m, and Trx f mutants were compared. All of the mutants are less efficient than wild-type Trx f in the activation of fructose-1,6-bisphosphatase and are altered in both S0.5 and Vmax. In contrast to literature reports, the activation of NADP-dependent malate dehydrogenase does not display rate saturation kinetics with respect to the concentration of Trx f, thereby signifying very weak interactions between the two proteins. The mutants of Trx f likewise interact only weakly with NADP-dependent malate dehydrogenase, but the apparent second-order rate constants for activation are increased compared to that with wild-type Trx f. Thus, Lys-58, Asn-74, Gln-75, and Asn-77 of Trx f contribute to its interaction with target enzymes and influence target protein selectivity.

Efficient expression of the gene for spinach phosphoribulokinase in Pichia pastoris and utilization of the recombinant enzyme to explore the role of regulatory cysteinyl residues by site-directed mutagenesis.

Phosphoribulokinase (PRK), unique to photosynthetic organisms, is regulated in higher plants by thioredoxin-mediated thiol-disulfide exchange in a light-dependent manner. Prior attempts to overexpress the higher plant PRK gene in Escherichia coli for structure-function studies have been hampered by sensitivity of the recombinant protein to proteolysis as well as toxic effects of the protein on the host. To overcome these impediments, we have spliced the spinach PRK coding sequence immediately downstream from the AOX1 (alcohol oxidase) promoter of Pichia pastoris, displacing the chromosomal AOX1 gene. The PRK gene is now expressed, in response to methanol, at 4-6% of total soluble protein, without significant in vivo degradation of the recombinant enzyme. This recombinant spinach PRK is purified to homogeneity by successive anion-exchange and dye-affinity chromatography and is shown to be electrophoretically and kinetically indistinguishable from the authentic spinach counterpart. Site-specific replacement of all of PRK's cysteinyl residues (both individually and in combination) demonstrates a modest catalytically facilitative role for Cys-55 (one of the regulatory residues) and the lack of any catalytic role for Cys-16 (the other regulatory residue), Cys-244, or Cys-250. Mutants with seryl substitutions at position 55 display non-hyperbolic kinetics relative to the concentration of ribulose 5-phosphate. Sulfate restores hyperbolic kinetics and enhances kinase activity, presumably reflecting conformational differences between the position 55 mutants and wild-type enzyme. Catalytic competence of the C16S-C55S double mutant proves that mere loss of free sulfhydryl groups by oxidative regulation cannot account entirely for the accompanying total inactivation.

The molecular pathway for the regulation of phosphoribulokinase by thioredoxin f.

Phosphoribulokinase (PRK) is one of several plant enzymes that is regulated by thiol-disulfide exchange as mediated by thioredoxin, which contains spatially vicinal, redox-active cysteinyl residues. In an earlier study (Brandes, H. K., Larimer, F. W., Geck, M. K., Stringer, C. D., Schürmann, P., and Hartman, F. C. (1993) J. Biol. Chem. 268, 18411-18414), our laboratory identified Cys-46 of thioredoxin f (Trx), as opposed to the other candidate Cys-49, as the primary nucleophile that attacks the disulfide of target proteins. The goal of the present study was to identify which of the two redox-active cysteinyl residues of PRK (Cys-16 or Cys-55) is paired with Cys-46 of Trx in the interprotein disulfide intermediate of the overall oxidation-reduction pathway. Incubation of a mixture of the C16S mutant of PRK and the C49S mutant of Trx with Cu2+ results in covalent complex formation as detected by SDS-polyacrylamide gel electrophoresis. Complexation is fully reversible by dithiothreitol and is retarded by ligands for PRK. Under the same conditions, Cu2+ induces very little complex formation between the following pairs of mutants: C16S PRK/C46S Trx, C55S PRK/C49S Trx, and C55S PRK/C46S Trx. When either 5-thio-2-nitrobenzoate-derivatized C16S or C55S PRK, as mimics of the oxidized (disulfide) form of the enzyme, is mixed with C49S Trx, stable covalent complex formation occurs only with the C16S PRK. Thus, two independent approaches identify Cys-55 of PRK in the intermolecular disulfide pairing with Trx.

Oxygenation mechanism of ribulose-bisphosphate carboxylase/oxygenase. Structure and origin of 2-carboxytetritol 1,4-bisphosphate, a novel O2-dependent side product generated by a site-directed mutant.

Site-directed mutagenesis has implicated active-site Lys329 of Rhodospirillum rubrum ribulose-1,5-bisphosphate carboxylase/oxygenase (Rubisco) in promoting the reaction of CO2 with the 2,3-enediol of ribulose bisphosphate and in stabilizing carboxylation intermediates [Hartman, F. C., & Lee, E. H. (1989) J. Biol. Chem. 264, 11784-11789; Lorimer, G. H., Chen, Y.-R., & Hartman, F. C. (1993) Biochemistry 32, 9018-9024]. Although the K329A mutant is greatly impaired in carboxylation, it catalyzes formation of the enediol, which is misprocessed to an O2-dependent side product [Harpel, M. R., & Hartman, F. C. (1994) Biochemistry 33, 5553-5561]. We now identify this novel side product as 2-carboxytetritol 1,4-bisphosphate (CTBP) by mass spectrometry, 1H-, 13C-, and 31P-NMR spectroscopy, and periodate oxidation. H2O2 accumulates during formation of CTBP, which we show to be derived from a transient precursor, the dicarbonyl D-glycero-2,3-pentodiulose 1,5-bisphosphate. The isolated dicarbonyl bisphosphate is processed by K329A to CTBP. These results, combined with isotope-labeling studies, suggest that CTBP arises by H2O2 elimination from an improperly stabilized peroxy adduct of the enediol intermediate, followed by rearrangement of the resulting dicarbonyl. Therefore, normal oxygenation, as catalyzed by wild-type Rubisco, is not a spontaneous reaction but must involve stabilization of the peroxy intermediate to mitigate formation of the dicarbonyl bisphosphate and subsequently CTBP. CTBP formation verifies the identity of Rubisco's previously invoked oxygenase intermediate, provides additional mechanistic insight into the oxygenation reaction, and shows that Lys329 promotes oxygenation as well as carboxylation. These results may be relevant to other oxygenases, which also exploit substrate carbanions rather than organic cofactors or transition metals for biological oxygen utilization.

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.

Mechanistic insights provided by deletion of a flexible loop at the active site of ribulose-1,5-bisphosphate carboxylase/oxygenase.

To evaluate the functions of flexible loop 6 at the active site of Rhodospirillum rubrum D-ribulose-1,5-bisphosphate carboxylase/oxygenase, the loop was truncated by cassette mutagenesis, whereby seven residues of the twelve-residue loop were excised and replaced by two glycyl residues. The purified loop-deletion mutant was totally devoid of carboxylase activity, but retained substantial catalytic competency in the enolization of ribulose bisphosphate (the initial step in the overall carboxylase pathway) and in normal processing of the six-carbon carboxylated intermediate (the terminal steps in the overall carboxylase pathway). Hence, catalytic impairment resides predominantly at the stage of carboxylation of the initial enediol(ate), a conclusion compatible with mechanistic deductions derived from crystallographic analyses. A critical role of loop 6 in the stabilization of the transition state for carboxylation is reinforced by the findings that the loop-deletion mutant displays preferentially compromised affinity for an analogue of the carboxylated intermediate relative to ribulose bisphosphate and that the mutant converts the substrate to a dicarbonyl compound as a consequence of beta-elimination of phosphate from the initial enediol(ate).

Chemical rescue by exogenous amines of a site-directed mutant of ribulose 1,5-bisphosphate carboxylase/oxygenase that lacks a key lysyl residue.

Ligand binding to ribulose 1,5-bisphosphate carboxylase/oxygenase immobilizes the flexible loop 6 of the beta/alpha barrel domain in its closed conformation. Lys329, located at the apex of this loop, interacts electrostatically with Glu48 of the adjacent subunit and with the CO2-derived carboxylate of the carboxylated reaction intermediate [Knight et al. (1990) J. Mol. Biol. 215, 113-160]. Previous studies have implicated Lys329 in the addition of CO2 to the initial enediol(ate) intermediate: mutants at position 329 catalyze enolization of ribulose 1,5-bisphosphate and processing of isolated carboxyketone intermediate, but are severely impaired in overall carboxylation and the tight-binding of the carboxylated intermediate analogue 2-carboxyarabinitol 1,5-bisphosphate. Using the chemical rescue method of Toney and Kirsch [(1989) Science 243, 1485-1488], we show that these defects are partially overcome by exogenous amines. For example, ethylamine enhances the carboxylation rate of K329A by about 80-fold and strengthens complexation of 2-carboxyarabinitol 1,5-bisphosphate. The CO2/O2 specificity of K329A is increased by amines, but remains lower than the wild-type value. Despite the pronounced enhancement of carboxylase activity, amines do not influence the rate at which ribulose 1,5-bisphosphate is enolized by K329A. Rescue of K329A follows an apparent Brønsted relationship with a beta of 1, implying complete protonation of amine in the rescued transition state. Rate saturation with respect to amine concentration and the different steric preferences for amines between K329A and K329C suggest that the amines bind to the enzyme in the position voided by the mutation.(ABSTRACT TRUNCATED AT 250 WORDS)

Beta-elimination of phosphate from reaction intermediates by site-directed mutants of ribulose-bisphosphate carboxylase/oxygenase.

Five residues (Thr-53, Asn-54, Gly-370, Gly-393, and Gly-394) of Rhodospirillum rubrum ribulose-bisphosphate carboxylase/oxygenase are positioned to serve as hydrogen-bond donors for the C1 phosphate of ribulose bisphosphate and thereby constrain conformational flexibility of the initial enediol(ate) intermediate (Knight, S., Andersson, I., and Brändén, C.-I. (1990) J. Mol. Biol. 215, 113-160). To study the functional contributions of the residues implicated in ribulose bisphosphate binding and intermediate stabilization, we have replaced them individually with alanine, either to remove the H-bonding group (T53A, N54A) or to introduce bulk (G370A, G393A, G394A). Consequences of substitutions include diminution of carboxylase activity (with a lesser impact on enolization activity), increase of Km (ribulose bisphosphate), and decrease of carboxylation: oxygenation specificity. During catalytic turnover of ribulose bisphosphate by several mutants, substantial amounts of the substrate are diverted to 1-deoxy-D-glycero-2,3-pentodiulose 5-phosphate, reflecting beta-elimination of phosphate from the enediol(ate) intermediate. This side product is not observed with wild-type enzyme, nor has it been reported with mutant enzymes characterized previously. Another consequence of disruption of the phosphate binding site is enhanced production of pyruvate, relative to wild-type enzyme, by some of the mutants due to decomposition of the acicarbanion of 3-phosphoglycerate (the terminal intermediate). These data provide direct evidence that phosphate ligands stabilize conformations of intermediates that favor productive turnover and mitigate beta-elimination at two stages of overall catalysis.

Perturbation of reaction-intermediate partitioning by a site-directed mutant of ribulose-bisphosphate carboxylase/oxygenase.

To explore the roles of active-site Glu48 of ribulose-bisphosphate carboxylase/oxygenase from Rhodospirillum rubrum, the E48Q mutant has been characterized with respect to kinetics and product distribution. Although the kcat for carboxylase activity is only 0.6% of the wild-type value, the mutant retains full activity in catalyzing the conversion of the carboxylated reaction intermediate to 3-phosphoglycerate and retains 10% of the normal activity in catalyzing the enolization of ribulose bisphosphate. Thus, the mutant is preferentially impaired in the carboxylation step. Partitioning of the enediol(ate) intermediate during turnover of ribulose bisphosphate is perturbed dramatically in the case of the mutant protein. Whereas the wild-type enzyme displays a CO2/O2 specificity factor of 11, the corresponding parameter of the mutant is only 0.3, thereby signifying a shift of the relative reactivity of the enediol(ate) in favor of O2. The mutant protein is also unable to protect the enediol(ate) against misprotonation with consequential conversion of ribulose bisphosphate to xylulose bisphosphate. This side reaction, undetected with wild-type R. rubrum enzyme, proceeds as rapidly as carboxylation of D-ribulose 1,5-bisphosphate by the E48Q mutant. Formation of xylulose bisphosphate by the mutant does not appear to account for the decline in carboxylase activity that occurs during the course of an assay. These studies demonstrate the multiple functionalities of Glu48 in the facilitation of catalysis and in directing intermediate partitioning in the preferred direction.

Direct identification of the primary nucleophile of thioredoxin f.

Thioredoxin, by virtue of the proximal active-site sulfhydryls (Trp-Cys-Gly-Pro-Cys), catalyzes thiol-disulfide exchange with specific target enzymes. Considerable data (chemical modification, spectroscopic, and crystallographic) have implicated the cysteinyl residue nearest the N terminus of thioredoxin as the primary nucleophile; however, direct proof has been lacking. Proof is now provided by characterization of site-directed mutants of thioredoxin f with respect to activation of chloroplastic fructose-1,6-bisphosphatase (FBPase). The C49S mutant retains the capacity to activate FBPase, whereas the C46S mutant is totally lacking in this regard. Based on kinetics of FBPase activation, wild-type and C49S thioredoxins exhibit half-saturation values of 0.9 and 4 microM, respectively. Lack of activation by C46S is not because of failure to interact with FBPase, for it exhibits a Ki of 5 microM in competition with wild-type thioredoxin. Therefore, in the normal thioredoxin-catalyzed reduction pathway, Cys-46 is the nucleophile required to attack the disulfide of the substrate and Cys-49 serves to cleave the mixed disulfide intermediate, thus allowing for the release of oxidized thioredoxin and the reduced target enzyme.