Adrenodoxin with a COOH-terminal deletion (des 116-128) exhibits enhanced activity.
Adrenodoxin, purified from bovine adrenal cortex, was subjected to trypsin cleavage to yield a trypsin-resistant form, designated TT-adrenodoxin. Sequencing with carboxypeptidase Y identified the trypsin cleavage site as Arg-115, while Edman degradation indicated no NH2-terminal cleavage. Native adrenodoxin and TT-adrenodoxin exhibited similar affinity for adrenodoxin reductase as determined in cytochrome c reductase assays. In side chain cleavage assays using cytochrome P-450scc, however, TT-adrenodoxin demonstrated greater activity than adrenodoxin with cholesterol, (22R)-22-hydroxycholesterol, or (20R,22R)-20,22-dihydroxycholesterol as substrate. This enhanced activity is due to increased affinity of TT-adrenodoxin for cytochrome P-450scc; TT-adrenodoxin exhibits a 3.8-fold lower apparent Km for the conversion of cholesterol to pregnenolone. TT-Adrenodoxin was also more effective in coupling with cytochrome P-450(11) beta, exhibiting a 3.5-fold lower apparent Km for the 11 beta-hydroxylation of deoxycorticosterone. In the presence of partially saturating cholesterol, TT-adrenodoxin elicited a type I spectral shift with cytochrome P-450scc similar to that induced by adrenodoxin, and spectral titrations showed that oxidized TT-adrenodoxin exhibited a 1.5-fold higher affinity for cytochrome P-450scc. These results establish that COOH-terminal residues 116-128 are not essential for the electron transfer activity of bovine adrenodoxin, and the differential effects of truncation at Arg-115 on interactions with adrenodoxin reductase and cytochromes P-450 suggest that the residues involved in the interactions are not identical.
Cupp
J R
JR
Department of Physiology and Biophysics, University of California, Irvine 92717.
Vickery
L E
LE
D000325:Adrenodoxin / Q000008:administration & dosage / Q000494:pharmacology*; D000595:Amino Acid Sequence; D000818:Animals; D002268:Carboxypeptidases / Q000378:metabolism; D002417:Cattle; D002784:Cholesterol / Q000378:metabolism; D003577:Cytochrome P-450 Enzyme System / Q000378:metabolism; D006888:Hydroxycholesterols / Q000378:metabolism; D007700:Kinetics; D008970:Molecular Weight; D009245:NADH Dehydrogenase / Q000378:metabolism; D010446:Peptide Fragments / Q000494:pharmacology*; D011284:Pregnenolone / Q000378:metabolism; D013329:Structure-Activity Relationship; D014357:Trypsin / Q000378:metabolism
D006888:Hydroxycholesterols; D010446:Peptide Fragments; D000325:Adrenodoxin; C003585:22-hydroxycholesterol; C003563:20,22-dihydroxycholesterol; D011284:Pregnenolone; D003577:Cytochrome P-450 Enzyme System; D002784:Cholesterol; D009245:NADH Dehydrogenase; D002268:Carboxypeptidases; C022633:serine carboxypeptidase; D014357:Trypsin