2 CONFORMATIONALLY VICINAL THIOLS AT THE ACTIVE-SITE OF LEISHMANIA-DONOVANI ADENOSINE KINASE

Inactivation of adenosine kinase (Adk) from Leishmania donovani correl ates with the modification of two conformationally vicinal cysteine re sidues. In contrast, Adk from hamster liver, despite being sensitive t o monothiol-blocking reagents, was insensitive to dithiol modifiers. I nactivation kinetics and substrate-protection studies along with doubl e-modification experiments successively with N-ethylmaleimide in the p resence of Ado and sodium m-arsenite-2,3-dimercaptopropanol or diazene dicarboxylic acid bis-N,N'-dimethylamide supported assignment of the t wo thiols at the Ado-binding site. Cystine bridge formation impaired t he 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, treatme nt of the enzyme with methylmethanethiosulphonate (MMTS) led to comple te inactivation without a marked change in protein fluorescence. Ado p rotected both fluorescence and catalytic activity against inactivation by both MMTS and dithiol-blocking reagents. Stern-Volmer quenching an alysis of the native and Ado-complexed enzyme suggested that, of the f our tryptophan residues, at least one is located at or near the active site. Furthermore quenching constants of native, Ado-complexed and di thiol-modified enzyme in the presence of either acrylamide or KI indic ated spatial proximity of tryptophan and two cysteine residues within the hydrophobic domain of the Ado-binding site. Taken together the res ults suggest important function(s) for the cysteine residue(s). A sche matic model is proposed to explain the inactivation of the enzyme by b oth monothiol- and dithiol-blocking reagents.