Gene/Protein Disease Symptom Drug Enzyme Compound
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Query: EC:3.4.21.1 (chymotrypsin)
 10,938 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

It is known that each subunit of the tetrameric flavocytochrome b2 can be cleaved by yeast proteases to fragments of molecular weight 33-36000 and 21 000, with some modification of catalytic properties, but without destruction of the oligomeric state of the protein. We report here experimental conditions which enabled us to simulate this specific cleavage in a controlled fashion with chymotrypsin and subtilisin. With trypsin and papain, on the other hand, it was not found possible to stop the digestion in such a way as to obtain a homogeneous still active product. A characterization of the enzymatic forms obtained by digestion with chymotrypsin and subtilisin at 0 degrees C shows that modification of enzymatic and solubility properties occurs in a stepwise fashion. It is also ccluded that cleavage by yeast proteases is accompanied by loss of 10 to 25 residues. At 37 degrees C, chymotrypsin digestion yields a heme-binding core of molecular weight 15 000, larger than the already characterized tryptic heme-binding core by about 40 residues. Although the latter is known to be very similar to trypsin-solubilized cytochrome b5, the lack of aggregation of the former in aqueous solution, its amino acid composition and circular dichroism spectra do not point to a similarity of its additional peptide segment with the hydrophobic tail of detergent-solubilized cytochrome b5.
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PMID:Controlled proteolysis of flavocytochrome b2. Characterization of a 15000-dalton heme-binding core and comparison with detergent solubilized cytochrome b5. 78 77

Each subunit of baker's yeast flavocytochrome b2 can be selectively cleaved by proteases into two fragments, amino-terminal fragment alpha and carboxy-terminal fragment beta. The primary structure of the former has been reported before [Ghrir, B., Becam, A. M. & Lederer, F. (1984) Eur. J. Biochem. 139, 59-74]. The amino acid sequence of the 197-residue fragment beta has now been established. The fragment was cleaved with cyanogen bromide; the three peptides thus obtained were submitted to digestions with Staphylococcus aureus V8 protease, chymotrypsin and trypsin, sometimes after succinylation. The complete fragment was also submitted to tryptic cleavage after citraconylation. Peptides were separated by thin-layer finger-printing or high-pressure liquid chromatography. They were mostly sequenced in a liquid-phase sequenator. The 511-residue amino acid sequence of the mature protein is thus completely established. Secondary structure predictions indicate an alternation of helical and extended structure, with a higher percentage of the former. Comparisons with other flavoproteins do not detect any significant sequence similarity.
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PMID:Complete amino acid sequence of flavocytochrome b2 from baker's yeast. 390 73

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.
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PMID:A cysteine cluster critical for flavin binding in flavocytochrome b2 from Baker's yeast. 633 38

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.
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PMID: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. 636 48

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.
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PMID:Study of a zone highly sensitive to proteases in flavocytochrome b2 from Saccharomyces cerevisiae. 703 12

Wild-type flavocytochrome b2 (L-lactate dehydrogenase) from Saccharomyces cerevisiae, as well as a number of its point mutants, can be expressed to a reasonable level as recombinant proteins in Escherichia coli (20-25 mg per liter culture) with a full complement of prosthetic groups. At the same expression level, active-site mutants Y254L and D282N, on the other hand, were obtained with an FMN/heme ratio significantly less than unity, which could not be raised by addition of free FMN. Evidence is provided that the flavin deficit is due to incomplete prosthetic group incorporation during biosynthesis. Flavin-free and holo-forms for both mutants could be separated on a Blue-Trisacryl M column. The far-UV CD spectra of the two forms of each mutant protein were very similar to one another and to that of the wild-type enzyme, suggesting the existence of only local conformational differences between the active holo-enzymes and the nonreconstitutable flavin-free forms. Selective proteolysis with chymotrypsin attacked the same bond for the two mutant holo-enzymes as in the wild-type one, in the protease-sensitive loop. In contrast, for the flavin-free forms of both mutants, cleavage occurred at more than a single bond. Identification of the cleaved bonds suggested that the structural differences between the mutant flavin-free and holo-forms are located mostly at the C-terminal end of the barrel, which carries the prosthetic group and the active site. Altogether, these findings suggest that the two mutations induce an alteration of the protein-folding process during biosynthesis in E. coli; as a result, the synchrony between folding and flavin insertion is lost. Finally, a preliminary kinetic characterization of the mutant holo-forms showed the Km value for lactate to be little affected; kcat values fell by a factor of about 70 for the D282N mutant and of more than 500 for the Y254L mutant, compared to the wild-type enzyme.
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PMID:On the lack of coordination between protein folding and flavin insertion in Escherichia coli for flavocytochrome b2 mutant forms Y254L and D282N. 766 48