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 [(PaPy(3))Mn(NO)](ClO(4)) (1) where PaPy(3)(-) 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 (50W tungsten lamp). With N(alpha)-benzoyl-l-arginine-p-nitroanilide (l-BApNA) as the substrate, the dissociation constant for the breakdown of the enzyme-inactivator complex (K(I)) and the overall inactivation rate constant (k(i)) were calculated to be 2.46mM and 64.8min(-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.
Afshar
Raman K
RK
Department of Chemistry and Biochemistry, University of California, Santa Cruz, CA 95064, USA.
Patra
Apurba K
AK
Mascharak
Pradip K
PK
D002851:Chromatography, High Pressure Liquid; D004791:Enzyme Inhibitors / Q000737:chemistry*; D008027:Light; D008345:Manganese / Q000737:chemistry*; D013058:Mass Spectrometry / Q000379:methods; D015394:Molecular Structure; D009569:Nitric Oxide / Q000737:chemistry; D009603:Nitroso Compounds / Q000737:chemistry*; D009942:Organometallic Compounds / Q000737:chemistry*; D010206:Papain / Q000737:chemistry* / Q000528:radiation effects*; D013997:Time Factors
D004791:Enzyme Inhibitors; D009603:Nitroso Compounds; D009942:Organometallic Compounds; D009569:Nitric Oxide; D008345:Manganese; D010206:Papain