Inhibition of Azotobacter vinelandii rhodanese by NO-donors.
Nitric oxide (NO) is a versatile regulatory molecule that affects enzymatic activity through chemical modification of reactive amino acid residues (e.g., Cys and Tyr) and by binding to metal centers. In the present study, the inhibitory effect of the NO-donors S-nitroso-glutathione (GSNO), (+/-)E-4-ethyl-2-[E-hydroxyimino]-5-nitro-3-hexenamide (NOR-3), and S-nitroso-N-acetyl-penicillamine (SNAP) on the catalytic activity of Azotobacter vinelandii rhodanese (RhdA) has been investigated. GSNO, NOR-3, and SNAP inhibit RhdA sulfurtransferase activity in a concentration- and time-dependent fashion. The absorption spectrum of the NOR-3-treated RhdA displays a maximum at 335 nm, indicating NO-mediated S-nitrosylation. RhdA inhibition by NO-donors correlates with S-nitrosothiol formation. The reducing agent dithiothreitol prevents RhdA inhibition by NO-donors, fully restores the catalytic activity, and reverts the NOR-3-induced RhdA absorption spectrum to that of the active enzyme. These results indicate that RhdA inhibition occurs via NO-mediated S-nitrosylation of the unique Cys230 catalytic residue.
Spallarossa
Andrea
A
Dipartimento di Scienze Farmaceutiche, Università di Genova, Via Benedetto XV 3, I-16132 Genoa, Italy.
Forlani
Fabio
F
Pagani
Silvia
S
Salvati
Luca
L
Visca
Paolo
P
Ascenzi
Paolo
P
Bolognesi
Martino
M
Bordo
Domenico
D
D016948:Azotobacter vinelandii / Q000201:enzymology*; D020134:Catalytic Domain; D003545:Cysteine / Q000737:chemistry; D004229:Dithiothreitol / Q000494:pharmacology; D004305:Dose-Response Relationship, Drug; D007700:Kinetics; D008958:Models, Molecular; D009569:Nitric Oxide / Q000378:metabolism; D020030:Nitric Oxide Donors / Q000494:pharmacology*; D009584:Nitrogen / Q000378:metabolism; D017434:Protein Structure, Tertiary; D011994:Recombinant Proteins / Q000378:metabolism; D013053:Spectrophotometry; D013696:Temperature; D013884:Thiosulfate Sulfurtransferase / Q000037:antagonists & inhibitors*; D013997:Time Factors
D020030:Nitric Oxide Donors; D011994:Recombinant Proteins; D009569:Nitric Oxide; D013884:Thiosulfate Sulfurtransferase; D003545:Cysteine; D009584:Nitrogen; D004229:Dithiothreitol