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F Lederer

Publications and source records attributed to F Lederer.

At least 127 records · Page 7Linked to original sources
PubMed1985

Methionyl-tRNA synthetase from Escherichia coli: primary structure at the binding site for the 3'-end of tRNAfMet.

It was previously shown that when the tryptic fragment of methionyl-tRNA synthetase from Escherichia coli is incubated with periodate-treated initiator tRNA, it is inactivated due to the formation of a covalent 1:1 complex that could be stabilized by reduction with cyanoborohydride [Hountondji, C., Fayat, G., & Blanquet, S. (1979) Eur. J. Biochem. 102, 247-250]. In this work, the residues labeled in the trypsin-modified enzyme have been identified. After chymotryptic digestion of the protein-tRNA complex, two major labeled peptides (A and B) and a minor one (C) were isolated and identified by sequencing. The radioactivity associated with peptides A-C represented 65-75, 20-25, and 2-4%, respectively, of the radioactivity eluted from the peptide maps. Peptides A and B encompassed lysines-335 and -61, respectively. Both these lysines were fully labeled. Peptide C encompassed lysines-142, -147, and -149, each of which was incompletely labeled. The significance of these results is discussed in light of the known crystallographic structure of the enzyme.

Amino Acid Sequence↗
PubMed1984

Baker's yeast flavocytochrome b2. A mechanistic study of the dehydrohalogenation reaction.

It has been shown that reduced flavocytochrome b2 not only catalyzes reduction of bromopyruvate [P. Urban, P.M. Alliel and F. Lederer (1983) Eur. J. Biochem. 134, 275-281] but also transforms it into pyruvate in a reductive elimination process. The dehydrohalogenation reaction also takes place when oxidized enzyme acts on bromolactate, but the reaction is more difficult to observe under these conditions because of its low efficiency compared to the normal oxidative process. The maximal rates of pyruvate production from bromopyruvate and chloropyruvate differ by a factor of less than 10, whereas elimination from fluoropyruvate cannot be detected. These results support a mechanism in which the dehydrohalogenation reaction takes place from a carbanion intermediate of the normal reductive-oxidative pathway.

Catalysis↗
PubMed1984

Primary structure of flavocytochrome b2 from baker's yeast. Purification by reverse-phase high-pressure liquid chromatography and sequencing of fragment alpha cyanogen bromide peptides.

Reverse-phase high-pressure liquid chromatography has been used for the purification of some large cyanogen bromide peptides from flavocytochrome b2 fragment alpha. Acetonitrile gradients at acid and/or neutral pH using mu Bondapak C18 columns were useful for the smaller peptides (43 and 67 residues). The two larger ones, alpha CB1 and alpha CB2, could only be separated from each other by trifluoroacetic acid/1-propanol gradients on mu Bondapak-CN columns. The various systems tested are presented and compared. The elucidation of the amino acid sequence of alpha CB2 (95 residues), alpha CB3 (67 residues) and alpha CB4 (43 residues) is described. The fragments were digested with trypsin, chymotrypsin and Staphylococcus aureus V8 protease as necessary. Fragment alpha CB2 was also cleaved at the unique tryptophanyl bond with cyanogen bromide. Peptides were fractionated by Sephadex chromatography, thin-layer finger-printing and/or high-pressure liquid chromatography. Peptides were sequenced mostly in the liquid phase sequenator. The cyanogen bromide peptides could be ordered using information obtained previously, as well as additional data obtained in this work. Together with the previous elucidation of cytochrome b2 core sequence and of the hinge region [Guiard, B. and Lederer, F. (1976) Biochimie (Paris) 58, 305--316; Ghrir, R. and Lederer, F. (1981) Eur. J. Biochem. 120, 279--287], the present results enable us to present the complete sequence of fragment alpha (314 residues) with only three overlaps missing between cyanogen bromide peptides. Sequence comparisons with other known flavoproteins do not indicate any noticeable similarity. Structural predictions indicate an alteration of alpha helices and beta structure. The possibility that the non-heme-binding portion of fragment alpha could constitute a flavin-binding domain is discussed.

Amino Acid Sequence↗
PubMed1983

On the transhydrogenase activity of baker's yeast flavocytochrome b2.

It is shown that, when baker's yeast flavocytochrome b2 is incubated with bromopyruvate in the presence of excess lactate, a transhydrogenation reaction takes place which produces bromolactate and pyruvate. The heme remains reduced during the reaction. It is further shown that reduced flavocytochrome b2 can catalyze the reduction of a number of other keto acids like pyruvate (the product of the physiological reaction) and other halogenopyruvates. Determinations of forward and reverse reaction rates, as well as of the redox potentials of the halogenolactate/halogenopyruvate couples lead to the conclusion that the transhydrogenation reaction is under thermodynamic control. Determinations of the steady-state deuterium isotope effect show that the rate-limiting step in the oxidation of halogenolactates is abstraction of the alpha-hydrogen (probably as a proton), as is the case for lactate itself. According to the principle of microscopic reversibility, the rate-limiting step in the reverse reaction must be protonation of the putative carbanion.

Heme↗
PubMed1983

Two homologous cytochromes b5 in a single cell.

The amino acid sequence of the heme-binding domains of rat liver cytochromes b5 from outer mitochondrial membranes and from microsomes has been determined by a combination of automatic and manual degradation of fragments generated by trypsin digestion and by cleavage at tryptophan. Tryptic peptides were separated by high-pressure liquid chromatography. The sequence of microsomal cytochrome b5 is identical with the one published by Ozols and Heinemann after completion of this study [Biochim. Biophys. Acta (1982) 704, 163-173]. The sequence of outer membrane cytochrome b5 differs from the microsomal one at 38 positions out of 91. There are 40 positions invariant between this sequence and the eight microsomal sequences published thus far. The non-conservative substitutions are located at the surface of the known three-dimensional structure of calf microsomal cytochrome b5 except for the substitution of histidine-15 by arginine. This paper brings the final proof that two iso-cytochromes b5 exist in the same cell. Their high degree of similarity as well as their differential cellular localization raise some questions which are briefly discussed.

Amino Acid Sequence↗
PubMed1983

A cysteine cluster critical for flavin binding in flavocytochrome b2 from Baker's yeast.

We have recently described the addition of 2-keto-3-butynoic acid to flavin-free flavocytochrome b2, a reaction which leads to the loss of flavin-binding capacity ('inactivation') [D. Pompon and F. Lederer (1982) Eur. J. Biochem. 129, 143-137]. For total inactivation, the extrapolated incorporation value was 0.9 mol reagent/mol subunit. In this work we report the results of sequence studies which elucidate the nature of the modification. The modified protein was cleaved with cyanogen bromide and the peptides separated on Sephadex G-100 and SP-Sephadex C-25. 14C-labeled peptides were digested with trypsin and chymotrypsin and smaller labeled fragments purified by chromatography on Sephadex G-50 and thin-layer fingerprinting. It is shown that three cysteine residues are fractionally labeled with nearly complete mutual exclusion. Furthermore, a fraction of the modified peptides is found under the form of cross-linked fragments, where two cysteines have added to the same ketobutynoate molecule. Only two of the possible cross-links were found. These results show that the three cysteines are close to one another in space in the flavin-free enzyme and hence probably also in the holoenzyme. These results, combined with those obtained in the affinity labeling reaction of holoenzyme with bromopyruvate [Alliel et al. (1982) Eur. J. Biochem. 122, 553-558], show that the three residues are located in or close to the active site. Their possible role is discussed.

Binding Sites↗
PubMed1983

On the presence of a heme-binding domain homologous to cytochrome b(5) in Neurospora crassa assimilatory nitrate reductase.

Assimilatory nitrate reductase has been purified with 55% recovery from a Neurospora crassa nmr-1 nit-6 mutant, using a modification of a published procedure. It possesses one heme per 240 000 g, and subunits of mol. wt. 68 000. Upon digestion with chymotrypsin, a heme-binding domain was isolated by gel filtration; its visible spectrum was highly similar to that of cytochrome b(5). On SDS gels, the fraction showed two heme-containing bands of 10 000 and 12 5000 daltons; their amino acid composition was not very different, suggesting that they originated from the same region of the polypeptide chain. After S-carboxymethylation, the mixture of bands was submitted to cyanogen bromide cleavage, and the fragments were separated by h.p.l.c. The two largest fragments yielded an identical sequence upon automated degradation. This sequence (39 residues with some gaps) could be easily aligned with that of cytochrome b(5) starting close to the N terminus. These results are discussed in terms of the possible quaternary structure of N. crassa nitrate reductase, whose heme-binding domain proves to be another member of the family of b(5)-like cytochromes.

Journal Article↗
PubMed1982

Reconstitution of liver NADH: cytochrome b5 oxidoreductase and of Desulfovibvio vulgaris flavodoxin with 1-carba-1-deazaflavin.

Flavin-free cytochrome b5 reductase was reconstituted with 1-deazaflavin and 5-deazaflavin mononucleotides and dinucleotides. The 5-deazaenzyme functioned in transhydrogenation reactions but lacked electron transferase activity. The 1-deazaenzyme was fully competent for both input and output reactions. The flavin reduction rate was lowered about sevenfold upon N-1 substitution of FAD, but hydrogen abstraction from NADH remained the limiting step. Autoxidation of the reduced enzyme was more rapid than with the normal cofactor. Oxidation was accompanied by appearance of a transient blue-type semiquinone and superoxide ion production. Flavin-free apoflavodoxin was reconstituted with 1-deaza-1-carbaflavin mononucleotide (1-deaza-FMN). Its behaviour toward dithionite and oxygen was qualitatively highly similar to that of native flavodoxin. These observations contrast with the fact that apoflavocytochrome b2 could not be reconstituted with 1-deaza-FMN [Pompon. D. and Lederer, F. (1979) Eur. J. Biochem. 96. 571-579]. These results, as well as other data from the literature, are discussed in the light of existing hypotheses, which try to correlate flavin protein interactions and flavoprotein function.

Anaerobiosis↗
PubMed1982

Bromopyruvate as an affinity label for Baker's yeast flavocytochrome b2. Identification of an active-site cysteine and characterization of some cysteine peptides.

It was previously reported that bromopyruvate behaves as an active-site-directed reagent for flavocytochrome b2 [Mulet and Lederer (1977) Eur. J. Biochem. 73, 443-447], but that some unspecific labeling also took place [Alliel, Mulet, and Lederer (1980) Eur. J. Biochem. 105, 343-351]. In this work, radioactive peptides were purified after labeling the enzyme with bromo[2-14C]pyruvate. Direct proteolysis of the labeled enzyme led to a multiplicity of labeled peptides, due to incomplete proteolysis. Four of them were characterized, corresponding to two unique cysteine residues. Cyanogen bromide cleavage of the labeled protein, followed by enzymatic digestion, led to the isolation of peptides corresponding to four cysteines, including the two previously identified ones. Comparison of the specific radioactivity of the various labeled peptides lead us to the conclusion that the active-site cysteine must be the one present in the 85-residue cyanogen bromide peptide alpha CB3. The sequence around that cysteine is Ala-Ser-Cys-Ser-Pro-Gln-Gln-Ile-Ile-Glu-Ala-Ala-.

Affinity Labels↗
PubMed1982

A residue critical for flavin binding in flavocytochrome b2 from Baker's yeast. Inactivation and labeling of flavin-free enzyme by 2-keto-3-butynoate.

The reagent 2-keto-3-butynoic acid is the product formed in the reaction between the suicide reagent 2-hydroxy-3-butynoate and a number of flavoproteins. We describe in this paper the inactivation of flavin-free flavocytochrome b2 by 2-keto-3-butynoate, in a rapid reaction which introduces 0.9 mol reagent for total inactivation. The modification results in loss of affinity for flavin and affects a cysteine residue. We also describe in this paper a simple enzymatic method for preparing 2-keto-3-butynoate, as well as some properties of the reagent, in particular its stability and susceptibility to nucleophilic attack. We show that at neutral pH it is highly specific for thiol compounds. Some properties of the adduct formed with glutathione are described. These experiments should pave the way for the use of 2-keto-3-butynoate with other proteins.

Binding Sites↗
PubMed1981

Study of a zone highly sensitive to proteases in flavocytochrome b2 from Saccharomyces cerevisiae.

Flavocytochrome b2 from baker's yeast is a bifunctional tetrameric protein which carries two prosthetic groups, FMN and heme, per subunit of Mr 58 000. The amino terminus of the subunit is wrapped around the heme and constitutes the so-called cytochrome b2 core (Mr 11 000), homologous to cytochrome b5. It has been shown in the past that a number of proteases (yeast proteases, chymotrypsin) preferentially cleave the peptide chain at a point situated much further down the polypeptide chain than the C terminus of the heme-binding domain. Some enzymatic parameters are concomitantly modified, but not the quaternary structure. This paper describes the conditions for selective proteolysis of intact flavocytochrome b2 and of its various previously studied stable nicked forms by the protease from Staphylococcus aureus V8. Successive attack by a combination of two proteases is also described. We have established the amino acid sequence of the area where proteolytic attack takes places, and shown that chymotrypsin and S. aureus protease open only one bond, whereas yeast proteases remove five residues from the central part. The various nicked forms, some of which have lost up to 16 amino acid residues, have been enzymatically characterized. These and previous results lend support to, but do not prove, the idea that the flavodehydrogenase part of flavocytochrome b2 may be composed of two domains, linked by the region accessible to proteases. That area might constitute a hinge or rather a clasp between the domains.

Amino Acid Sequence↗
PubMed1981

Amino-acid sequence of the cytochrome c from the yeast Hansenula anomala. Identification of three methylated positions.

The cytochrome c of the yeast Hansenula anomala has been purified and sequenced. A combination of automatic and manual sequencing methods was used on the whole protein and on fragments obtained by cyanogen bromide cleavage and proteolytic fragmentation. The cytochrome presents an amino-terminal extension of six residues and the C-terminal one-residue deletion typical of plant and fungal cytochrome c. Lysines 72 and 73 are trimethylated, lysine 55 partly monomethylated and partly dimethylated. Positions 73 and 55 have never been found methylated before.

Amino Acid Sequence↗
PubMed1980

Flavocytochrome b2 (Baker's yeast). Deuterium isotope effect studied by rapid-kinetic methods as a probe for the mechanism of electron transfer.

The use of DL-[2-2H]lactate in steady-state measurements of ferricyanide reduction by flavocytochrome b2 at 30 degrees C has previously yielded an isotope effect of 5 [F. Lederer (1974) Eur. J. Biochem. 46, 393--399]. We report here studies carried out at 5 degrees C with L-[2-2H]lactate, where flavin and heme reduction were observed in the stopped-flow apparatus, in the absence of acceptor. The generally biphasic reduction curves were analysed according to a new mathematical treatment which allowed us to derive microscopic constants from initial reduction rates. It has thus been possible to determine an isotope effect of 8 on flavin reduction, 6 on heme reduction, compared to 4 in the steady state. Consequently, two slightly rate-limiting steps occur after the first one where the alpha-hydrogen is abstracted. It has also been possible to calculate the substrate association and dissociation rate constants for intact enzyme. The studies were carried out in parallel on intact and cleaved cytochrome b2. The results suggest that proteolysis affects essentially the steps involved in flavin reduction, and not intramolecular electron transfer steps. Moreover, the experimental data obtained at low rates of electron entry have led us to reexamine a previously proposed scheme for electron transfer [Capeillère-Blandin, Bray, Iwatsubo and Labeyrie (1975) Eur. J. Biochem. 54, 549--566]. An alternative model based on computer-simulation studies will be presented in a paper in this journal.

Deuterium↗
PubMed1980

Bromopyruvate as an affinity label for baker's yeast flavocytochrome b2. Stoichiometry of incorporation and localization on the peptide chain.

We have reported in a previous communication a kinetic study showing bromopyruvate to behave as an active-site-directed reagent for flavocytochrome b2. It is shown here that inactivation is accompanied by incorporation of 3 mol reagent/subunit of oxidized intact enzyme and 4 mol reagent/subunit nicked enzyme. Only one of the modifications is presumed to be responsible for activity loss. All labeled groups are found to be cysteines. Incubation of reduced nicked enzyme with bromopyruvate results in total protection of activity and loss of only one sulfhydryl group. A subsequent incubation in the presence of the competitive inhibitor sulfite leads to some more loss of non-essential groups. After these two pretreatments, incubation in the presence of bromo[2-14C]pyruvate results in incorporation of 1.2--1.5 mol reagent/subunit concomitant with the loss of about 0.8 active site. A study of the distribution of label between fragments alpha and beta has been carried out using gel electrophoresis and Sephadex filtration after selective proteolysis. It is shown that the active-site sulfhydryl group corresponds to one of the four cysteines situated in the last two thirds of fragment alpha. The structural and functional implications of these results is discussed.

Affinity Labels↗
PubMed1980

Binding of Cibacron blue F3GA to the flavin and NADH sites in cytochrome b5 reductase.

The behaviour of cytochrome b5 reductase holoenzyme and apoenzyme toward blue-dextran--Sepharose has been studied. Holoenzyme was adsorbed at low ionic strength and could be eluted with 100 microM NADH or NAD+. Flavin-free enzyme was even more strongly bound and could be eluted with 1 M NaCl, or 100 microM NADH + 10 microM FAD. Separately the cofactors were without effect. FMN was less effective than FAD. ADP and AMP eluted nothing. Cibacron blue F3GA was found to exert a mixed inhibition on NADH oxidation. Dye binding to holoenzyme elicited a characteristic red shift in its spectrum. Comparison of the difference spectrum amplitude at 680 and 585 nm showed the presence of a second binding mode at higher dye concentrations. These results point to the existence for cytochrome b5 reductase of two binding sites with high affinity for blue-dextran--Sepharose: the NADH binding site and flavin binding site. For the latter it is clear that isoalloxazine pocket must play a role in dye binding. Cytochrome b5 reductase is the second flavoenzyme which has been shown to have affinity for immobilized dye at the flavin site, the first one being flavocytochrome b2, and FMN-dependent enzyme [D. Pompon and F. Lederer (1978) Eur. J. Biochem. 90, 563--569].

Animals↗
PubMed1979

Amino acid sequence of the 'b5-like' heme-binding domain from chicken sulfite oxidase.

We present in this paper the sequence of the heme-binding domain of chicken sulfite oxidase which can be obtained by chymotryptic digestion of the native enzyme. The results of an automatic degradation have been reported previously. In the present work peptides were obtained from the heme-binding domain by digestion with trypsin, chymotrypsin and Staphylococcus aureus V8 protease; they were manually sequenced by the dansyl/Edman procedure. The evidence thus obtained is sufficient to completely establish the order of the 97 residues. In addition, two rounds of Edman degradation on sulfite oxidase itself allowed us to identify the same two residues, H-Ala-Pro, present at the N-terminus of the heme-binding domain; this result suggests that the latter constitutes the amino-terminal end of the sulfite oxidase peptide chain. The data presented here confirm the strong similarity between sulfite oxidase and microsomal cytochrome b5 already suggested by our first results. A sequence alignment is proposed for the two proteins. Inspection of the calf liver cytochrome b5 three-dimensional model together with the alignment suggests a similar overall structure for sulfite oxidase core with a limited number of backbone modifications. Our results point to a common evolutionary origin for sulfite oxidase core and microsomal cytochrome b5.

Amino Acid Sequence↗