Phosphoribulokinase: isolation and sequence determination of the cysteine-containing active-site peptide modified by 5′-p-fluorosulfonylbenzoyladenosine

Krieger, Timothy J.; Mende‐Mueller, Liane; Miziorko, Henry M.

doi:10.1016/0167-4838(87)90130-0

Cited by 38 publications

(23 citation statements)

References 20 publications

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“…Polyclonal antibodies to the purified protein were used to isolate a Chiamydomonas cDNA clone, which, upon sequencing, was found to contain the entire coding region. (6,11,21), and its location is consistent with results of affinity labeling of the enzyme with an ATP analog (1 1). Also, the roles of several metabolic effectors in regulation of spinach phosphoribulokinase have been determined (5).…”

supporting

confidence: 63%

“…The cysteine residues involved in thioredoxin-mediated regulation ofthe spinach enzyme have been identified (21). A nucleotidebinding consensus sequence has been noted (6,11,21), and its location is consistent with results of affinity labeling of the enzyme with an ATP analog (1 1). Also, the roles of several metabolic effectors in regulation of spinach phosphoribulokinase have been determined (5).…”

mentioning

confidence: 63%

“…In contrast, the amino-terminus is well conserved and is known to be essential for function. In addition to the regulatory cysteine located at position 16, a nucleotide-binding consensus sequence S-G-X-G-K-S-T (7) was previously located in spinach phosphoribulokinase (6,11,21), and is identical in the Chlamydomonas enzyme. Other regions of striking homology are apparent, including 23 consecutive residues (positions 229-251 in the consensus sequence) which are common to both sequences.…”

Section: Discussionmentioning

confidence: 99%

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Chlamydomonas reinhardtii Phosphoribulokinase

Roesler¹,

Ogren²

1990

Plant Physiol.

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The sequence and kinetic properties of phosphoribulokinase purified from Chiamydomonas reinhardtii were determined and compared with the spinach (Spinacea oleracea) enzyme. Chiamydomonas phosphoribulokinase was purified to apparent homogeneity, with a specific activity of 410 micromoles per minute per milligram. Polyclonal antibodies to the purified protein were used to isolate a Chiamydomonas cDNA clone, which, upon sequencing, was found to contain the entire coding region. (6,11,21), and its location is consistent with results of affinity labeling of the enzyme with an ATP analog (1 1). Also, the roles of several metabolic effectors in regulation of spinach phosphoribulokinase have been determined (5). Investigations of phosphoribulokinase of several prokaryotic organisms have shown that the bacterial enzyme differs markedly from the spinach enzyme with respect to kinetic parameters, subunit size and number, pH optima, activation by thiols, and regulation by metabolites (1,15,26,29).In contrast to the knowledge of the higher plant and bacterial enzymes, little biochemical characterization and no sequence data have been reported for algal phosphoribulokinase. To gain insight into structure-function relationships of this enzyme, the sequence and kinetic parameters of phosphoribulokinase from the green alga Chlamydomonas reinhardtii were determined and compared with corresponding information for the spinach enzyme. MATERIALS AND METHODS Strain and Culture ConditionsChlamydomonas reinhardtii strain 21 37A+ was maintained on an acetate medium which was described previously (28

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supporting

confidence: 63%

mentioning

confidence: 63%

Section: Discussionmentioning

confidence: 99%

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Chlamydomonas reinhardtii Phosphoribulokinase

Roesler¹,

Ogren²

1990

Plant Physiol.

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“…Cys-16 (like Cys-32 of E. coli thioredoxin) displays an abnormally low pK a (22) and hyperreactivity toward numerous sulfhydryl reagents (23)(24)(25), whereas Cys-55 (like Cys-35 of E. coli thioredoxin) is far less accessible for chemical modification (26 -28). Apparently, the sulfur atom of Cys-55 is either more exposed in the disulfide form of PRK than in the free sulfhydryl form or the interaction of Trx with PRK induces a conformational change that renders this sulfur susceptible to attack by Cys-46 of Trx.…”

Section: Resultsmentioning

confidence: 99%

The Molecular Pathway for the Regulation of Phosphoribulokinase by Thioredoxin f

Brandes

Larimer

Hartman

1996

Journal of Biological Chemistry

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Thioredoxins are small (12-14 kDa) ubiquitous proteins that control the redox state of diverse target proteins by the mediation of thiol-disulfide exchanges (1, 2). The redox-active sulfhydryls of thioredoxin are located in the highly conserved active-site sequence -Trp-Cys-Gly-Pro-Cys-. The pathway for the reduction of a protein disulfide by thioredoxin entails nucleophilic attack by one of the active-site sulfhydryls to form a protein-protein mixed disulfide followed by intramolecular displacement of the reduced target protein with concomitant formation of oxidized thioredoxin (Fig. 1). As the mixed disulfide is disfavored thermodynamically, its direct characterization has not been possible. However, the active-site cysteinyl residue of thioredoxin that is nearest the N terminus (Cys-32 of the Escherichia coli protein) is well established as the primary nucleophile. Originally, this assignment was deduced from the high chemical reactivity and the low pK a (6.7) of Cys-32 (3-6) and was subsequently supported by the three-dimensional structure of E. coli thioredoxin, in which Cys-32 is solventaccessible and Cys-35 is inaccessible (7). More recently, Cys-32 of E. coli thioredoxin, the corresponding residue of human thioredoxin, and the corresponding residue of the closely related protein glutaredoxin were shown to engage in mixed disulfide bond formation with low molecular weight thiols or peptides (8 -11). Finally, the C49S mutant of chloroplastic thioredoxin f (Trx) 1 retains the capacity to activate chloroplastic fructose-1,6-bisphosphatase, whereas the C46S mutant is totally inactive in this regard. Thus, Cys-46 of Trx (analogous to Cys-32 of E. coli thioredoxin) is verified as the primary nucleophile in the reduction of in vivo target proteins, thereby minimizing the possibility that protein-protein interactions might alter the relative accessibility and reactivity of the active-site sulfhydryls of thioredoxin.In contrast to the rigorous proof that Cys-46 of Trx participates in intermolecular disulfide bond formation, the identity of the pairing residue in any target protein has not been established heretofore. We have addressed this issue with PRK by examining the potential of site-directed mutants, which lack either one of the two redox-active sulfhydryls (Cys-16 or Cys-55), to form stable mixed disulfides with the C49S mutant of Trx. This approach, which should be applicable to other target proteins, clearly identifies Cys-55 as the participant in the intermolecular mixed disulfide. EXPERIMENTAL PROCEDURESMaterials-The following chemicals and biologicals were procured from the indicated vendors: Bicine and DTT, Research Organics, Inc.; components for kinase assays, Sigma; cupric sulfate, Fisher Scientific; DTNB, Pierce. D-Ribulose 5-phosphate was prepared from D-ribose 5-phosphate by the action of phosphoribose isomerase, and its concen-

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“…No domain have been identified through recognition of a nucleotide-binding consensus sequence (11,21) and by affinity labeling the spinach enzyme with ATP analogs (1 1, 16). The reductive/oxidative regulation of phosphoribulokinase also is not clearly understood.…”

mentioning

confidence: 99%