Light-induced inhibition of papain by a {Mn-NO}6 nitrosyl: Identification of papain-SNO adduct by mass spectrometry

Modification of Cys25 at the active site of the cysteine protease papain by S-nitrosylation inhibits its hydrolytic ability. Previous studies have demonstrated that NO donors N-nitrosoanilines inhibit papain activity via formation of S-NO bond formation at the active site while NO donors such as S-nitroso-N-acetyl-penicillamine (SNAP), N-nitrosoaniline derivatives, and S-nitroso-glutathione (GSNO) inhibit the enzyme via S-thiolation by thiyl radicals generated from the S-nitrosothiols. In this study, we report papain inactivation by a photosensitive {Mn-NO}6 nitrosyl [(PaPy3)Mn(NO)](ClO4) (1) where PaPy3- is the anion of the designed ligand N,N-bis(2-pyridylmethyl)amine-N-ethyl-2-pyridine-2-carboxamide. This nitrosyl releases NO upon exposure to visible light of low intensity (50 W tungsten lamp). With Nα-benzoyl-l-arginine-p-nitroanilide (l-BApNA) as the substrate, the dissociation constant for the breakdown of the enzyme-inactivator complex (KI) and the overall inactivation rate constant (ki) were calculated to be 2.46 mM and 64.8 min−1, respectively. The papainS-NO adduct has been identified using electrospray mass spectrometry (ESI-MS). The results demonstrate that controlled inactivation of papain can be achieved with the {Mn-NO}6 nitrosyl 1 and light. The reaction is clean and the extent of inactivation is directly proportional to the exposure time.