Exceptional sensitivity of Rubisco activase to thermal denaturation in vitro and in vivo.

Heat stress inhibits photosynthesis by reducing the activation of Rubisco by Rubisco activase. To determine if loss of activase function is caused by protein denaturation, the thermal stability of activase was examined in vitro and in vivo and compared with the stabilities of two other soluble chloroplast proteins. Isolated activase exhibited a temperature optimum for ATP hydrolysis of 44 degrees C compared with > or =60 degrees C for carboxylation by Rubisco. Light scattering showed that unfolding/aggregation occurred at 45 degrees C and 37 degrees C for activase in the presence and absence of ATPgammaS, respectively, and at 65 degrees C for Rubisco. Addition of chemically denatured rhodanese to heat-treated activase trapped partially folded activase in an insoluble complex at treatment temperatures that were similar to those that caused increased light scattering and loss of activity. To examine thermal stability in vivo, heat-treated tobacco (Nicotiana rustica cv Pulmila) protoplasts and chloroplasts were lysed with detergent in the presence of rhodanese and the amount of target protein that aggregated was determined by immunoblotting. The results of these experiments showed that thermal denaturation of activase in vivo occurred at temperatures similar to those that denatured isolated activase and far below those required to denature Rubisco or phosphoribulokinase. Edman degradation analysis of aggregated proteins from tobacco and pea (Pisum sativum cv "Little Marvel") chloroplasts showed that activase was the major protein that denatured in response to heat stress. Thus, loss of activase activity during heat stress is caused by an exceptional sensitivity of the protein to thermal denaturation and is responsible, in part, for deactivation of Rubisco.

Crystal structure of the NADP(H)-dependent ketose reductase from Bemisia argentifolii at 2.3 A resolution.

Polyhydric alcohols are widely found in nature and can be accumulated to high concentrations as a protection against a variety of environmental stresses. It is only recently, however, that these molecules have been shown to be active in protection against heat stress, specifically in the use of sorbitol by the silverleaf whitefly, Bemisia argentifolii. We have determined the structure of the enzyme responsible for production of sorbitol in Bemisia argentifolii, NADP(H)-dependent ketose reductase (BaKR), to 2.3 A resolution. The structure was solved by multiwavelength anomalous diffraction (MAD) using the anomalous scattering from two zinc atoms bound in the structure, and was refined to an R factor of 21.9 % (R(free)=25.1 %). BaKR belongs to the medium-chain dehydrogenase family and its structure is the first for the sorbitol dehydrogenase branch of this family. The enzyme is tetrameric, with the monomer having a very similar fold to the alcohol dehydrogenases (ADHs). Although the structure determined is for the apo form, a phosphate ion in the active site marks the likely position for the adenyl phosphate of NADP(H). The catalytic zinc ion is tetrahedrally coordinated to Cys41, His66, Glu67 and a water molecule, in a modification of the zinc site usually found in ADHs. This modified zinc site seems likely to be a conserved feature of the sorbitol dehydrogenase sub-family. Comparisons with other members of the ADH family have also enabled us to model a ternary complex of the enzyme, and suggest how structural differences may influence coenzyme binding and substrate specificity in the reduction of fructose to sorbitol.

Isobemisiose: an unusual trisaccharide abundant in the silverleaf whitefly, Bemisia argentifolii.

The major soluble carbohydrates in the silverleaf whitefly, Bemisia argentifolii, were glucose, alpha,alpha-trehalose and an unknown sugar. Analysis of the unknown sugar and its chemical and enzymatic digestion products by high-performance liquid chromatography (HPLC) showed that it was probably a trisaccharide, consisting entirely of glucose, and containing both alpha,alpha-trehalose and isomaltose moieties. Matrix-assisted laser desorption mass spectrometry, mass spectrometry and 13C and 1H nuclear magnetic resonance spectroscopy confirmed that the sugar was a trisaccharide with the following structure: O-alpha-D-glucopyranosyl-(1-->6)-O-alpha-D-glucopyranosyl-(1<-->1)-alpha-D-glucopyranoside. This trisaccharide, found primarily in the bodies of B. argentifolii and not in their honeydew, is structurally similar to bemisiose [O-alpha-D-glucopyranosyl-(1-->4)-O-alpha-D-glucopyranosyl-(1<-->1)-alpha-D-glucopyranoside], a sugar first identified in Bemisia honeydew. Consequently, the common name isobemisiose is proposed for the newly identified sugar. Isobemisiose, which has not been previously reported to occur in nature, constituted as much as 46% (w/w) of the ethanol-soluble sugars in adult B. argentifolii, equivalent to approximately 10% of their dry weight. It was also found in similar quantities in immature B. argentifolii. Isobemisiose was detected in two other whitefly species and in several species of aphids, but at lesser concentrations than in B. argentifolii. Labeling and pulse-chase experiments using [14C]sucrose supplied to B. argentifolii in an artificial diet revealed that label accumulated in and was chased from isobemisiose more slowly than for either glucose or trehalose. Incubation of isobemisiose with cell-free extracts of B. argentifolii demonstrated that these whiteflies contained the necessary complement of enzymes to fully degrade isobemisiose to glucose. These labeling and digestion experiments indicate that isobemisose is probably a storage carbohydrate in B. argentifolii.

Heat stress induces the synthesis of a new form of ribulose-1,5-bisphosphate carboxylase/oxygenase activase in cotton leaves.

Ribulose-1,5-bisphosphate carboxylase/oxygenase (Rubisco; EC 4.1.1.39) activase mRNA and protein synthesis were measured in the leaves of cotton (Gossypium hirsutum L.) plants under control (28 degrees C) or heat-stress (41 degrees C) conditions. A decline in activase transcript abundance occurred rapidly during the photoperiod and was unaffected by heat stress. In response to high temperature, de novo protein synthesis rapidly shifted from mainly expression of Rubisco large and small subunits to the major heat-shock proteins, while de novo synthesis of the constitutively expressed 47- and 43-kDa activase polypeptides was not appreciably altered. However, heat stress induced the synthesis of a 46-kDa polypeptide that immunoprecipitated with antibodies monospecific to activase. Expression of the 46-kDa polypeptide ceased within 1 h of the return of heat-stressed plants to control conditions. Activase precursors of 55 and 51 kDa were detected among the in vitro translation products of RNA from control and heat-stressed plants. In addition, a 53-kDa polypeptide that also immunoprecipitated with anti-activase IgG was among the in vitro translation products of RNA from heat-stressed plants. This putative activase precursor did not occur among the in vitro translation products of RNA from plants that had recovered from heat stress. The levels of the constitutive 47- and 43-kDa activase polypeptides were similar in control and heat-stressed plants, based on immunoblotting with antibodies to activase. However, a 46-kDa cross-reacting polypeptide was also present in heat-stressed plants and constituted about 5% of the total activase after 48 h at high temperature. The identity of the heat-induced 46-kDa polypeptide as activase was confirmed by protein sequencing, which showed that its N-terminal sequence was identical to that of the constitutive 47-kDa activase polypeptide. The presence of multiple isoforms for both the 47- and 43-kDa activase polypeptides on immunoblots of two-dimensional gels and the complex banding pattern on Southern blots together suggest the existence of more than one activase gene and the possibility that the synthesis of the heat-induced activase polypeptide may be regulated transcriptionally. Induction of a new form of activase may constitute a mechanism of photosynthetic acclimation to heat stress in cotton.

Rubisco activase constrains the photosynthetic potential of leaves at high temperature and CO2.

Net photosynthesis (Pn) is inhibited by moderate heat stress. To elucidate the mechanism of inhibition, we examined the effects of temperature on gas exchange and ribulose 1,5-bisphosphate carboxylase/oxygenase (Rubisco) activation in cotton and tobacco leaves and compared the responses to those of the isolated enzymes. Depending on the CO(2) concentration, Pn decreased when temperatures exceeded 35-40 degrees C. This response was inconsistent with the response predicted from the properties of fully activated Rubisco. Rubisco deactivated in leaves when temperature was increased and also in response to high CO(2) or low O(2). The decrease in Rubisco activation occurred when leaf temperatures exceeded 35 degrees C, whereas the activities of isolated activase and Rubisco were highest at 42 degrees C and >50 degrees C, respectively. In the absence of activase, isolated Rubisco deactivated under catalytic conditions and the rate of deactivation increased with temperature but not with CO(2). The ability of activase to maintain or promote Rubisco activation in vitro also decreased with temperature but was not affected by CO(2). Increasing the activase/Rubisco ratio reduced Rubisco deactivation at higher temperatures. The results indicate that, as temperature increases, the rate of Rubisco deactivation exceeds the capacity of activase to promote activation. The decrease in Rubisco activation that occurred in leaves at high CO(2) was not caused by a faster rate of deactivation, but by reduced activase activity possibly in response to unfavorable ATP/ADP ratios. When adjustments were made for changes in activation state, the kinetic properties of Rubisco predicted the response of Pn at high temperature and CO(2).

Molecular basis for thermoprotection in Bemisia: structural differences between whitefly ketose reductase and other medium-chain dehydrogenases/reductases.

The silverleaf whitefly (Bemisia argentifolii, Bellows and Perring) accumulates sorbitol as a thermoprotectant in response to elevated temperature. Sorbitol synthesis in this insect is catalyzed by an unconventional ketose reductase (KR) that uses NADPH to reduce fructose. A cDNA encoding the NADPH-KR from adult B. argentifolii was cloned and sequenced to determine the primary structure of this enzyme. The cDNA encoded a protein of 352 amino acids with a calculated molecular mass of 38.2 kDa. The deduced amino acid sequence of the cDNA shared 60% identity with sheep NAD(+)-dependent sorbitol dehydrogenase (SDH). Residues in SDH involved in substrate binding were conserved in the whitefly NADPH-KR. An important structural difference between the whitefly NADPH-KR and NAD(+)-SDHs occurred in the nucleotide-binding site. The Asp residue that coordinates the adenosyl ribose hydroxyls in NAD(+)-dependent dehydrogenases (including NAD(+)-SDH), was replaced by an Ala in the whitefly NADPH-KR. The whitefly NADPH-KR also contained two neutral to Arg substitutions within four residues of the Asp to Ala substitution. Molecular modeling indicated that addition of the Arg residues and loss of the Asp decreased the electric potential of the adenosine ribose-binding pocket, creating an environment favorable for NADPH-binding. Because of the ability to use NADPH, the whitefly NADPH-KR synthesizes sorbitol under physiological conditions, unlike NAD(+)-SDHs, which function in sorbitol catabolism.

Effect of high temperature on the metabolic processes affecting sorbitol synthesis in the silverleaf whitefly, Bemisia argentifolii.

Whiteflies accumulate the polyhydric alcohol, sorbitol, when exposed to temperatures greater than about 30 degrees C. Feeding experiments using artificial diets containing labeled sucrose showed that more of the label was incorporated into whitefly bodies and less was excreted in the honeydew when feeding was conducted at 41 compared with 25 degrees C. Analysis of the components of the honeydew showed that more of the excreted label was in glucose and fructose and less in trehalulose at 41 degrees C than at 25 degrees C. A similar effect of temperature on honeydew composition occurred for whiteflies feeding on cotton leaves. Measurement of the activities of glycolytic, pentose-phosphate and polyol pathway enzymes at 30 and 42 degrees C showed that NADPH-dependent ketose reductase/sorbitol dehydrogenase (NADPH-KR/SDH), sucrase, glucokinase and glucose-6-phosphate dehydrogenase activities were stimulated to a greater extent at 42 degrees C than trehalulose synthase and fructokinase. NAD(+)-sorbitol dehydrogenase (NAD(+)-SDH) activity was inhibited at 42 degrees C. We propose that high temperature alters metabolic activity in a way that increases the availability of fructose and stimulates pentose-phosphate pathway activity, providing both the substrate and coenzyme for sorbitol synthesis. High temperature also increases the activity of NADPH-KR/SDH, the enzyme in whiteflies that synthesizes sorbitol, but inhibits the activity of NAD(+)-SDH, the enzyme that degrades sorbitol.

Involvement of two aspartate residues of Rubisco activase in coordination of the ATP gamma-phosphate and subunit cooperativity.

Aspartate residues are involved in coordination of the nucleotide-metal of several nucleotide triphosphatases. To examine interactions between Rubisco activase and ATP, site-directed mutations were made at two species-invariant aspartate residues, D174 and D231. In the absence of the magnesium cofactor, the mutant proteins D231R, D174Q, and D174A, but not D174E, bound ATP with higher affinity than did wild-type. In the presence of Mg2+, the affinity for ATP of D231R was further increased, but was reduced with mutations at D174. Although all mutants bound ATP, only D174E aggregated in response to ATP/Mg2+ and retained partial ATPase and Rubisco activation activities. In mixing experiments, the catalytically competent D174E stimulated wild-type ATPase activity, whereas the mutants lacking ATPase activity were inhibitory to wild-type enzyme and prevented aggregation. These results are consistent with a mechanism for activase that involves ATP-binding, subunit aggregation and ATP hydrolysis as sequential steps in the catalytic mechanism. The results also indicated that precise coordination of the gamma-phosphate is required for aggregation and depends on D174 and D231. To account for the pronounced cooperativity of Rubisco activase subunits, we suggest that coordination of the ATP gamma-phosphate may involve participation of residues from adjacent subunits.

A thermoprotective role for sorbitol in the silverleaf whitefly, Bemisia argentifolii.

Accumulation of polyols in insects is well known as a cold-hardening response related to overwintering or to protection against cold shock. The silverleaf whitefly (Bemisia argentifolii, Bellows and Perring) is a major insect pest in tropical and subtropical regions where heat stress and desiccation pose formidable threats to survival. We found that sorbitol levels increased ten-fold when whiteflies were exposed to elevated temperatures. Sorbitol levels rose from 0.16nmolwhitefly(-1) at 25 degrees C to 1.59nmolwhitefly(-1) at 42 degrees C. Sorbitol levels fluctuated diurnally under glasshouse and field conditions increasing ten-fold from morning to early afternoon. Feeding experiments on artificial diets showed that both temperature and dietary sucrose concentration were key factors influencing sorbitol accumulation. Cell free extracts prepared from adult whiteflies catalyzed NADPH-dependent fructose reduction, but were unable to reduce glucose with either NADPH or NADH. Radiotracer experiments with labeled glucose and fructose showed that fructose was the immediate precursor of sorbitol. Thus, sorbitol synthesis in the whitefly is apparently unconventional, involving conversion of fructose by a novel NADPH-dependent ketose reductase. We propose that sorbitol accumulation is a mechanism for thermoprotection and osmoregulation in the silverleaf whitefly, allowing the insect to thrive in environments conducive to thermal and osmotic stress.

The Two Forms of Ribulose-1,5-Bisphosphate Carboxylase/Oxygenase Activase Differ in Sensitivity to Elevated Temperature.

Ribulose-1,5-bisphosphate carboxylase/oxygenase activase often consists of two polypeptides that arise from alternative splicing of pre-mRNA. In this study recombinant versions of the spinach (Spinacea oleracea L.) 45- and 41-kD forms of activase were analyzed for their response to temperature. The temperature optimum for ATP hydrolysis by the 45-kD form was 45[deg]C, approximately 13[deg]C higher than the 41-kD form. When the two forms were mixed, the temperature response of the hybrid enzyme was similar to the 45-kD form. In the absence of adenine nucleotide, preincubation of either activase form at temperatures above 25[deg}C inactivated ATPase activity. Adenosine 5[prime]-([gamma]-thio)triphosphate, but not ADP, significantly enhanced the thermostability of the 45-kD form but was much less effective for the 41-kD form. Intrinsic fluorescence showed that the adenosine 5[prime]-([gamma]-thio)triphosphate-induced subunit aggregation was lost at a much lower temperature for the 41-kD than for the 45-kD form. However, the two activase forms were equally susceptible to limited proteolysis after heat treatment. The results indicate that (a) the 45-kD form is more thermostable than, and confers increased thermal stability to, the 41-kD form, and (b) a loss of subunit interactions, rather than enzyme denaturation, appears to be the initial cause of temperature inactivation of activase.

Physical and Kinetic Evidence for an Association between Sucrose-Phosphate Synthase and Sucrose-Phosphate Phosphatase.

The possible formation of a multienzyme complex between sucrose (Suc)-phosphate synthase (SPS) and Suc-phosphate phosphatase (SPP) was examined by measuring the rates of Suc-6-phosphate (Suc-6-P) synthesis and hydrolysis in mixing experiments with partially purified enzymes from spinach (Spinacia oleracea) and rice (Oryza sativa) leaves. The addition of SPP to SPS stimulated the rate of Suc-6-P synthesis. SPS inhibited the hydrolysis of exogenous Suc-6-P by SPP when added in the absence of its substrate (i.e. UDP-glucose) but stimulated SPP activity when the SPS substrates were present and used to generate Suc-6-P directly in the reaction. Results from isotope-dilution experiments suggest that Suc-6-P was channeled between SPS and SPP. A portion of the SPS activity comigrated with SPP during native polyacrylamide gel electrophoresis, providing physical evidence for an enzyme-enzyme interaction. Taken together, these results strongly suggest that SPS and SPP associate to form a multienzyme complex.

Activation of ribulose-1,5-biphosphate carboxylase/oxygenase (Rubisco) involves Rubisco activase Trp16.

The role of the N-terminal region of tobacco Rubisco activase in ATP hydrolysis and ribulose-1,5-bisphosphate carboxylase/oxygenase (Rubisco) activation was examined by construction of mutant proteins. Deletion of the first 50 amino acids of Rubisco activase almost completely eliminated the ability to activate Rubisco, without changing the ATP-hydrolyzing and self-associating properties of the enzyme. Thus, the N-terminus of Rubisco activase is distinct from the ATP-hydrolyzing domain and is required for Rubisco activation. Directed mutagenesis of the species-invariant tryptophan residue at position 16 inhibited Rubisco activation but not the binding or hydrolysis of ATP. The ability to activate Rubisco was less severely inhibited when Trp was replaced by a Tyr or Phe than by an Ala or Cys, indicating that an aromatic residue at position 16 and particularly a Trp is required for proper activation of Rubisco. Fluorescence quenching of the 7-nitrobenz-2-oxa-1,3-diazole-modified W16C mutant upon addition of nucleotide suggested that position 16 becomes more solvent accessible in response to nucleotide binding. However, changes in the intrinsic fluorescence of truncated and Trp16 mutants upon addition of ATP were similar to those of the wild type, evidence that Trp16 is not the residue reporting the conformational change that accompanies subunit association.

The mechanism of Rubisco activase: Insights from studies of the properties and structure of the enzyme.

Rubisco, the primary carboxylating enzyme in photosynthesis, must be activated to catalyze CO2 fixation. The concept of an 'activase', a specific protein for activating Rubisco, was first introduced in 1985 based largely on biochemical and genetic studies of a high CO2-requiring mutant of Arabidopsis (Salvucci et al. (1985) Photosynth Res 7: 193-201). Over the past ten years, details about the occurrence, structure, and properties of Rubisco activase have been elucidated. However, the mechanism of action of Rubisco activase remains elusive. This review discusses the need for and function of Rubisco activase and summarizes information about the properties and structure of Rubisco activase. The information is evaluated in the context of the mechanism of Rubisco activase.

Purification and Properties of a Unique Nucleotide Pyrophosphatase/Phosphodiesterase I That Accumulates in Soybean Leaves in Response to Fruit Removal.

Several unique proteins accumulate in soybean (Glycine max) leaves when the developing fruits are removed. In the present study, elevated levels of nucleotide pyrophosphatase and phosphodiesterase I activities were present in leaves of defruited soybean plants. The soluble enzyme catalyzing these reactions was purified nearly 1000-fold, producing a preparation that contained a single 72-kD polypeptide. The molecular mass of the holoenzyme was approximately 560 kD, indicating that the native enzyme was likely octameric. The purified enzyme hydrolyzed nucleotide-sugars, nucleotide di- and triphosphates, thymidine monophosphate p-nitrophenol, and inorganic pyrophosphate but not nucleotide monophosphates, sugar mono- and bisphosphates, or NADH. The subunit and holoenzyme molecular masses and the preference for substrates distinguish the soybean leaf nucleotide pyrophosphatase/phosphodiesterase I from other plant nucleotide pyrophosphatase/phosphodiesterase I enzymes. Also, the N-terminal sequence of the soybean leaf enzyme exhibited no similarity to the mammalian nucleotide pyrophosphatase/phosphodiesterase I, soybean vegetative storage proteins, or other entries in the data bank. Thus, the soybean leaf nucleotide pyrophosphatase/phosphodiesterase I appears to be a heretofore undescribed protein that is physically and enzymatically distinct from nucleotide pyrophosphatase/phosphodiesterase I from other sources, as well as from other phosphohydrolytic enzymes that accumulate in soybean leaves in response to fruit removal.

Rubisco, rubisco activase and ribulose-5-phosphate kinase gene expression and polypeptide accumulation in a tobacco mutant defective in chloroplast protein synthesis.

Expression of the genes for ribulose-1,5-bisphosphate carboxylase/oxygenase (Rubisco; rbcS and rbcL), Rubisco activase (rca) and ribulose-5-phosphate (Ru5-P) kinase (prk) and accumulation of the polypeptides was examined in chlorophyllous and chlorotic sectors of the DP1 mutant of Nicotiana tabacum. Plastids from chlorotic sectors of this variegated plastome mutant contained 30S and 50S ribosomal subunits, but had abnormally low levels of plastid polysomes. Consequently, mutant plastids were translationally repressed, unable to synthesize plastid-encoded polypeptides including the large subunit of Rubisco despite the presence of the corresponding mRNAs. Transcripts of rbcS accumulated to near wild type levels in chlorotic sectors, but there was little accumulation of the Rubisco small subunit (SS) polypeptide or holoenzyme. Messenger-RNA isolated from chlorotic sectors effectively directed the synthesis of Rubisco SS in vitro suggesting that posttranslational factors were responsible for the decrease in Rubisco SS abundance. Transcripts of rca and prk also accumulated to near wild type levels in chlorotic sectors and a diurnal rhythm in the abundance of rca mRNA was detected in green and chlorotic sectors. Despite the low abundance of Rubisco holoenzyme in chlorotic sectors, Rubisco activase and Ru5-P kinase polypeptides accumulated to significant levels. Activities of Rubisco and Ru5-P kinase paralleled protein levels, indicating that active forms of these enzymes were present in chlorotic sectors. The data indicate that the developmental events governing the accumulation of Rubisco activase and Ru5-P kinase polypeptides and the diurnal regulation of rca expression were not dependent on the attainment of photosynthetically competent plastids or the accumulation of Rubisco.

Photoaffinity labeling of the ATP binding domain of Rubisco activase and a separate domain involved in the activation of ribulose-1,5-bisphosphate carboxylase/oxygenase.

Photoaffinity labeling of Rubisco activase with 2- and 8-N3ATP was used to identify the adenine binding domain for ATP. Rubisco activase hydrolyzed both of these analogs of ATP and used their hydrolysis to support a low rate of Rubisco activation. When irradiated with ultraviolet light, these and other azido-substituted adenine nucleotides covalently modified Rubisco activase at two distinct binding sites. Competition binding experiments with ATP and ADP showed that one of the sites was the ATP binding domain. The other site was not a nucleotide binding domain per se but would bind adenine nucleotides if an azido moiety was present on the base. Tryptophan and other indoles prevented azidoadenine nucleotides from labeling this domain but afforded little protection to the ATP binding domain. The ability to selectively protect each of the two binding sites made it possible to localize the adenine binding domain for ATP to the region of Rubisco activase from N68-D74 and the other binding domain to a region near the N-terminus from Q10 to D14. Modification of the region from Q10 to D14 by photoaffinity labeling prevented Rubisco activase from promoting activation of Rubisco without affecting ATP hydrolysis. These data suggest that a specific region of Rubisco activase near the N-terminus may be a site of interaction with Rubisco. Binding of azidoadenine nucleotides in this region appears to be fortuitous and may involve base-stacking with the species-invariant Trp at position 16 and hydrogen bonding of the azido moiety.

Site-directed mutagenesis of a reactive lysyl residue (Lys-247) of Rubisco activase.

Chemical modification of tobacco leaf ribulose-1,5-bisphosphate carboxylase/oxygenase (Rubisco) activase with water-soluble N-hydroxysuccinimide esters identified Lys-247 as a particularly reactive residue necessary for maximal catalytic activity [M.E. Salvucci (1993) Plant Physiol. 103, 501-508]. To further explore the role of Lys-247 in catalysis, this species-invariant residue of Rubisco activase was changed to Arg, Cys, and Gln by mutagenesis of a cDNA clone of the mature form of the tobacco enzyme. Analysis of the purified recombinant proteins showed that all three point mutations reduced the rate of ATP hydrolysis to 2 to 3% of the wild-type enzyme and completely abolished the ability of Rubisco activase to promote activation of decarbamylated Rubisco. Replacement of Lys-247 with Arg, Cys, or Gln had a comparatively minor effect on ATP binding, but eliminated the increase in ATPase-specific activity that normally occurs with increasing concentrations of Rubisco activase protein. In mixing experiments, the K247R mutant enzyme inhibited Rubisco activation by wild-type Rubisco activase, indicating that interactions between Rubisco and Rubisco activase were disrupted by even the most conservative of the substitutions. Chemical elaboration of the K247C mutant by treatment with 2-bromoethylamine converted 39% of the thiols at position 247 to the aminoethyl derivative, but failed to improve the catalytic performance of the mutant enzyme. Our results indicate that the requirement for a lysyl residue at position 247 of Rubisco activase is very stringent, consistent with its proposed role in coordinating precise interactions with gamma-phosphate of ATP.

Identification of the major regulatory phosphorylation site in sucrose-phosphate synthase.

Sucrose-phosphate synthase (SPS; EC 2.4.1.14) is regulated in part by reversible protein phosphorylation. When dephospho-SPS is partially purified from illuminated spinach leaves and incubated with [gamma-32P]ATP the enzyme is phosphorylated by a copurifying protein kinase. In this report, 32P-phosphopeptides from tryptic digests of in vitro phosphorylated SPS were purified by metal-ion affinity chromatography and reversed-phase high-performance liquid chromatography. Three distinct 32P-phosphopeptides were resolved. Edman sequencing of the major phosphopeptide (which contained > 80% of the total 32P) identified the amino acid sequence as Ile-Ser-Ser(P)-Val-Glu-Met-Met-Asp-Asn-Trp-Ala-Asn-Thr-Phe-Lys. This sequence corresponds to residues 156 to 170 of the deduced amino acid sequence of spinach SPS [Klein, R. R., Crafts-Brandner, S. J., and Salvucci, M. E. (1993) Planta 190, 498-510, and Sonnewald, U., Quick, W. P., MacRae, E., Krause, K.-P., and Stitt, M. (1993) Planta 189, 174-181]. Identification of the phosphoseryl residue was accomplished by manual Edman sequencing. The two other phosphopeptides, which each contained less than 10% of the total 32P, were not sequenced. An Escherichia coli expressed, 26-kDa fragment of SPS which contains the major phosphorylation site was a substrate for the protein kinase which copurifies with SPS. Two-dimensional peptide mapping analysis of this fragment showed the major phosphopeptide was present but not the other site(s), suggesting that other peptides are derived from a site other than Ser158. These results provide additional indirect evidence for the presence of multiple phosphorylation sites in SPS.

Covalent modification of a highly reactive and essential lysine residue of ribulose-1,5-bisphosphate carboxylase/oxygenase activase.

Chemical modification of ribulose-1,5-bisphosphate carboxylase/oxygenase (Rubisco) activase with water-soluble N-hydroxysuccinimide esters was used to identify a reactive lysyl residue that is essential for activity. Incubation of Rubisco activase with sulfosuccinimidyl-7-amino-4-methylcoumarin-3-acetate (AMCA-sulfo-NHS) or sulfosuccinimidyl-acetate (sulfo-NHS-acetate) caused progressive inactivation of ATPase activity and concomitant loss of the ability to activate Rubisco. AMCA-sulfo-NHS was the more potent inactivator of Rubisco activase, exhibiting a second-order rate constant for inactivation of 239 M-1 s-1 compared to 21 M-1 s-1 for sulfo-NHS-acetate. Inactivation of enzyme activity by AMCA-sulfo-NHS correlated with the incorporation of 1.9 mol of AMCA per mol of 42-kD Rubisco activase monomer. ADP, a competitive inhibitor of Rubisco activase, afforded considerable protection against inactivation of Rubisco activase and decreased the amount of AMCA incorporated into the Rubisco activase monomer. Sequence analysis of the major labeled peptide from AMCA-sulfo-NHS-modified enzyme showed that the primary site of modification was lysine-247 (K247) in the tetrapeptide methionine-glutamic acid-lysine-phenylalanine. Upon complete inactivation of ATPase activity, modification of K247 accounted for 1 mol of AMCA incorporated per mol of Rubisco activase monomer. Photoaffinity labeling of AMCA-sulfo-NHS- and sulfo-NHS-acetate-modified Rubisco activase with ATP analogs derivatized on either the adenine base or on the gamma-phosphate showed that K247 is not essential for the binding of adenine nucleotides per se. Instead, the data indicated that the essentiality of K247 is probably due to an involvement of this highly reactive, species-invariant residue in an obligatory interaction that occurs between the protein and the nucleotide phosphate during catalysis.