Resonance Energy Transfer Determination of the Distance between the Four Cysteine-364 Residues in Saccharomyces cerevisiae Phosphoenolpyruvate Carboxykinase

Alvear, Marysol; Encinas, M. V.; Herrera, Lisandra; Cardemil, Emilio

doi:10.1006/abbi.1994.1107

Cited by 2 publications

(7 citation statements)

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“…C458S enzyme. As shown in Table 2, enzyme inactivation was effectively prevented by the combined presence of saturating concentrations of ADP plus MnCla, in agreement with previous results (Rojas et al, 1993;Alvear et al, 1994).…”

Section: Methodssupporting

confidence: 92%

“…by several sulfhydryl-directed reagents in the presence of MnADP (Alvear et al, 1992(Alvear et al, , 1994Rojas et al, 1993)-it is reasonable to conclude that Cys365 and Cys458 are spatially near the nucleotide binding site. Chemical modification of these residues would inhibit the access or binding of substrates to the catalytic site.…”

Section: Methodsmentioning

confidence: 99%

“…Selected fractions were collected and concentrated under a N2 stream at 30 °C, and each was rechromatographed on a reverse-phase Supelco C4 column, which was eluted under the same conditions. The purified fractions were subjected to amino acid sequence analysis as previously described (Roías et al, 1993;Alvear et al, 1994).…”

Section: Methodsmentioning

confidence: 99%

“…On the basis of chemical modification experiments, it had been inferred that this enzyme contains reactive cysteinyl residues in the substrate binding region. In previous research we have shown that the fluorescent reagents 1,5-I-AEDANS and IANBD specifically react with Cys365(2 ' (Alvear et al, 1992(Alvear et al, , 1994, while pyrenyliodoacetamide labels Cys365 and CyS458(2) (R0jas et al, 1993). In the latter case, close proximity of the reactive residues has been deduced from the observation of pyrene excimer emission in the protein labeled with 2 mol of probe per mole of enzyme subunit.…”

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confidence: 99%

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Saccharomyces cerevisiae Phosphoenolpyruvate Carboxykinase: Revised Amino Acid Sequence, Site-Directed Mutagenesis, and Microenvironment Characteristics of Cysteines 365 and 458

Krautwurst

Encinas²,

Marcus³

et al. 1995

Biochemistry

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Two cysteine residues in phosphoenolpyruvate (PEP) carboxykinase from Saccharomyces cerevisiae [ATP:oxaloacetate carboxy-lyase (transphosphorylating), EC 4.1.1.49] the modification of which leads to enzyme inactivation have been subjected to site-directed mutagenesis. PEP carboxykinase is inactivated by alkylation of Cys365 or Cys458; however, mutation of either or both of these residues to serine has little effect on the enzymatic activity. These results eliminate any possible catalytic function for these cysteinyl residues. In the course of this work, discrepancies in the published nucleotide sequence of the S. cerevisiae PEP carboxykinase gene were detected that alter the deduced amino acid sequence. Several of these discrepancies were verified through the sequencing of proteolytic peptides. Our results indicate that the protein corresponds to a 549 amino acid polypeptide and that the positions previously assigned to Cys364 and Cys457 correspond to Cys365 and Cys458. The individual reactivities and the microenvironment characteristics around these sulfhydryl groups were investigated by their selective modification with the fluorescent reagent N-(1-pyrenyl)maleimide (PyM). Our findings indicate that Cys458 is 7-fold more reactive toward the sulfhydryl-directed probe than Cys365, while quenching experiments of PyM-labeled mutant enzymes suggest that the former residue is located in a region more accessible to water than the latter.

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Section: Methodssupporting

confidence: 92%

Section: Methodsmentioning

confidence: 99%

Section: Methodsmentioning

confidence: 99%

mentioning

confidence: 99%

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Saccharomyces cerevisiae Phosphoenolpyruvate Carboxykinase: Revised Amino Acid Sequence, Site-Directed Mutagenesis, and Microenvironment Characteristics of Cysteines 365 and 458

Krautwurst

Encinas²,

Marcus³

et al. 1995

Biochemistry

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“…We chose to utilize the IAEDANS–IANBD pair since these two dyes have been used successfully in previous FRET studies (for examples, see refs 58 and 59). The relatively short R 0 value [34 Å measured for the free dyes (data not shown)] was considered appropriate for the resolution needed to map the binding site on a large protein like SecA [~100 Å in its longest dimension (34)].…”

Section: Resultsmentioning

confidence: 99%

Mapping of the Signal Peptide-Binding Domain of Escherichia coli SecA Using Förster Resonance Energy Transfer

et al. 2010

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Identification of the signal peptide-binding domain within SecA ATPase is an important goal for understanding the molecular basis of SecA preprotein recognition as well as elucidating the chemomechanical cycle of this nanomotor during protein translocation. In this study, Förster resonance energy transfer methodology was employed to map the location of the SecA signal peptide-binding domain using a collection of functional monocysteine SecA mutants and alkaline phosphatase signal peptides labeled with appropriate donor-acceptor fluorophores. Fluorescence anisotropy measurements yielded an equilibrium binding constant of 1.4 or 10.7 μM for the alkaline phosphatase signal peptide labeled at residue 22 or 2, respectively, with SecA, and a binding stoichiometry of one signal peptide bound per SecA monomer. Binding affinity measurements performed with a monomerbiased mutant indicate that the signal peptide binds equally well to SecA monomer or dimer. Distance measurements determined for 13 SecA mutants show that the SecA signal peptide-binding domain encompasses a portion of the preprotein cross-linking domain but also includes regions of nucleotidebinding domain 1 and particularly the helical scaffold domain. The identified region lies at a multidomain interface within the heart of SecA, surrounded by and potentially responsive to domains important for binding nucleotide, mature portions of the preprotein, and the SecYEG channel. Our FRET-mapped binding domain, in contrast to the domain identified by NMR spectroscopy, includes the two-helix finger that has been shown to interact with the preprotein during translocation and lies at the entrance to the protein-conducting channel in the recently determined SecA-SecYEG structure.Proteins are secreted across or integrated into biological membranes by means of a variety of protein translocation systems that have been characterized over the past several decades. In Escherichia coli, the major pathway for protein secretion is the general secretion (Sec) pathway that is composed of two fundamental components: the SecYEG heterotrimeric complex that comprises the protein-conducting channel and the SecA ATPase nanomotor that drives † This work was supported by Grants GM42033 and GM37639 from Figure S1), binding affinity of unlabeled SP2 and IANBD-labeled SP2 with IAEDANS-labeled SecA-Cys-256 ( Figure S2) and mapping of the FRET-determined signal peptide-binding site on the SecA crystal structures of B. subtilis, E. coli, T. thermophilus, and Mycobacterium tuberculosis ( Figure S3). This material is available free of charge via the Internet at http://pubs.acs.org. NIH Public Access Author ManuscriptBiochemistry. Author manuscript; available in PMC 2010 April 7. Recently, a model for preprotein translocation has been proposed on the basis of a SecASecYEG cocrystal structure (10) and disulfide cross-linking studies (11). In this model, SecA captures the preprotein in a clamp formed by nucleotide-binding domain 2 (NBD-2) 1 , the preprotein binding domain (PPXD), and the h...

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Resonance Energy Transfer Determination of the Distance between the Four Cysteine-364 Residues in Saccharomyces cerevisiae Phosphoenolpyruvate Carboxykinase

Cited by 2 publications

References 0 publications

Saccharomyces cerevisiae Phosphoenolpyruvate Carboxykinase: Revised Amino Acid Sequence, Site-Directed Mutagenesis, and Microenvironment Characteristics of Cysteines 365 and 458

Saccharomyces cerevisiae Phosphoenolpyruvate Carboxykinase: Revised Amino Acid Sequence, Site-Directed Mutagenesis, and Microenvironment Characteristics of Cysteines 365 and 458

Mapping of the Signal Peptide-Binding Domain of Escherichia coli SecA Using Förster Resonance Energy Transfer

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