Studies on the flavin-binding region of old yellow enzyme with an active site probe, 8-fluoro-8-demethyl FMN.
The brewer's yeast old yellow enzyme (OYE) was reconstituted with 8-fluoro-8-demethyl FMN (8F-FMN). The reconstituted enzyme exhibited absorption maxima at 355 and 450 nm in the visible region. This reconstituted enzyme underwent no further spectral changes, showing no evidence of modification in the flavin moiety. However, when the reconstituted enzyme was subjected to specific limited proteolysis with bovine alpha-chymotrypsin, gradual spectral changes were observed with disappearance of the 355- and 450-nm bands accompanied by the appearance of a new band at 496 nm. Identical spectral changes were observed when the proteolytically cleaved OYE (nicked OYE) was reconstituted with 8F-FMN. The process associated with these spectral changes was found to be unimolecular by kinetic analysis. Reverse-phase HPLC analysis revealed that these spectral changes resulted from covalent bond formation between 8F-FMN and the protein moiety after the proteolytic cleavage of the protein into 14K and 34K fragments. The reverse-phase HPLC monitored at 490 nm showed that the chromophore with 496 nm absorption maximum was covalently attached to the 14K fragment. The amino acid sequence analysis of the flavinylated 14K fragment together with that of the 14K fragment of native OYE indicated that the N-terminal leucine of the 14K fragment is the site of flavinylation. These findings imply that the amino group of the N-terminal leucine of the 14K fragment became available as the result of proteolysis and that this amino group nucleophilically attacked the 8-position of 8F-FMN, forming a covalent bond between the flavin moiety and the 14K fragment.(ABSTRACT TRUNCATED AT 250 WORDS)
Fujii
S
S
Laboratory of Chemistry, Kansai Medical University, Osaka.
Kuroda
K
K
Kasai
S
S
Miura
R
R
D000595:Amino Acid Sequence; D001051:Apoenzymes / Q000378:metabolism; D001665:Binding Sites; D005415:Flavins; D015335:Molecular Probes; D008969:Molecular Sequence Data; D008970:Molecular Weight; D009252:NADPH Dehydrogenase / Q000737:chemistry / Q000378:metabolism*; D010446:Peptide Fragments / Q000737:chemistry / Q000302:isolation & purification / Q000378:metabolism; D012256:Riboflavin / Q000031:analogs & derivatives; D012441:Saccharomyces cerevisiae / Q000201:enzymology; D013053:Spectrophotometry
D001051:Apoenzymes; D005415:Flavins; D015335:Molecular Probes; D010446:Peptide Fragments; C037198:8-fluoro-8-demethylriboflavin; D009252:NADPH Dehydrogenase; D012256:Riboflavin