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.
D000595:Amino Acid Sequence; D001665:Binding Sites; D002918:Chymotrypsin / Q000494:pharmacology; D007770:L-Lactate Dehydrogenase / Q000378:metabolism*; D042844:L-Lactate Dehydrogenase (Cytochrome); D010446:Peptide Fragments; D010447:Peptide Hydrolases / Q000494:pharmacology; D011487:Protein Conformation / Q000187:drug effects; D012441:Saccharomyces cerevisiae / Q000201:enzymology*; D013211:Staphylococcus aureus / Q000201:enzymology
D010446:Peptide Fragments; D007770:L-Lactate Dehydrogenase; D042844:L-Lactate Dehydrogenase (Cytochrome); D010447:Peptide Hydrolases; D002918:Chymotrypsin