Inactivation of adenosine kinase (Adk) from Leishmania donovani correlates with the modification of two conformationally vicinal cysteine residues. In contrast, Adk from hamster liver, despite being sensitive to monothiol-blocking reagents, was insensitive to dithiol modifiers. Inactivation kinetics and substrate-protection studies along with double-modification experiments successively with N-ethylmaleimide in the presence of Ado and sodium m-arsenite–2,3-dimercaptopropanol or diazenedicarboxylic acid bis-N,N´-dimethylamide supported assignment of the two thiols at the Ado-binding site. Cystine bridge formation impaired the ability of the modified enzyme to bind to the substrate. Tryptophan fluorescence of the enzyme was quenched after modification by dithiol-blocking reagents with concomitant loss of activity. However, treatment of the enzyme with methylmethanethiosulphonate (MMTS) led to complete inactivation without a marked change in protein fluorescence. Ado protected both fluorescence and catalytic activity against inactivation by both MMTS and dithiol-blocking reagents. Stern–Volmer quenching analysis of the native and Ado-complexed enzyme suggested that, of the four tryptophan residues, at least one is located at or near the active site. Furthermore quenching constants of native, Ado-complexed and dithiol-modified enzyme in the presence of either acrylamide or KI indicated spatial proximity of tryptophan and two cysteine residues within the hydrophobic domain of the Ado-binding site. Taken together the results suggest important function(s) for the cysteine residue(s). A schematic model is proposed to explain the inactivation of the enzyme by both monothiol- and dithiol-blocking reagents.
Mammalian Spermidine Synthase—Identification of Cysteine Residues and Investigation of the Putrescine Binding Site—
