On the structure of old yellow enzyme studied by specific limited proteolysis.
Limited proteolysis of brewer's yeast old yellow enzyme (OYE) was carried out with bovine pancreatic alpha-chymotrypsin. The reaction proceeded with a decrease of the NADPH oxidase activity, generating specifically two peptides (designated as 34K and 14K fragments) with apparent molecular weights of 34,000 and 14,000, respectively. The same proteolytic treatment of apo OYE resulted in rapid and complete digestion of the protein. The 34K and 14K fragments are so intimately associated with each other that the isolation of each peptide from the other in the native form was unsuccessful. However, the complex of the two fragments was separated from the intact OYE and termed "nicked OYE." Nicked OYE still retained FMN and showed a visible-absorption spectrum slightly modified from that of intact OYE. Nicked OYE showed decreased affinity toward rho-bromophenol as compared to intact OYE. Nicked OYE exhibited lower Km and Vmax values than intact OYE in the NADPH oxidase reaction. The 34K and 14K fragments could be separated from each other by reversed-phase HPLC under denaturing conditions and the amino acid sequences of the two fragments and intact OYE in the amino terminal regions were determined. The N-terminal sequence of the 34K fragment coincided with that of intact OYE, indicating that the 34K fragment lies in the N-terminal side of OYE. The N-terminal sequence of the 14K fragment was found to show homology with the site of flavodoxin where it forms an electron-transfer complex with cytochrome c. The characteristic feature of this region is the presence of acidic residues and is shared by the FMN domain of NADPH-cytochrome P-450 reductase. We interpret these findings as indicating that OYE has a physiological role as an electron transfer component.
D000595:Amino Acid Sequence; D002851:Chromatography, High Pressure Liquid; D002918:Chymotrypsin / Q000378:metabolism; D004220:Disulfides / Q000378:metabolism; D004591:Electrophoresis, Polyacrylamide Gel; D005486:Flavin Mononucleotide / Q000378:metabolism; D007700:Kinetics; D008969:Molecular Sequence Data; D008970:Molecular Weight; D009247:NADH, NADPH Oxidoreductases / Q000378:metabolism; D009252:NADPH Dehydrogenase / Q000378:metabolism; D019255:NADPH Oxidases; D009251:NADPH-Ferrihemoprotein Reductase; D010446:Peptide Fragments / Q000302:isolation & purification / Q000378:metabolism; D011487:Protein Conformation; D012441:Saccharomyces cerevisiae / Q000201:enzymology*; D013053:Spectrophotometry
D004220:Disulfides; D010446:Peptide Fragments; D005486:Flavin Mononucleotide; D009247:NADH, NADPH Oxidoreductases; D009251:NADPH-Ferrihemoprotein Reductase; D019255:NADPH Oxidases; D009252:NADPH Dehydrogenase; D002918:Chymotrypsin