title: Two conformationally vicinal thiols at the active site of Leishmania donovani adenosine kinase.
abstract: 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.
authors.affiliation: Leishmania Group, Indian Institute of Chemical Biology, Calcutta, India.
authors.forename: T K
authors.initials: TK
authors.lastname: Bagui
authors.forename: M
authors.initials: M
authors.lastname: Ghosh
authors.forename: A K
authors.initials: AK
authors.lastname: Datta
mesh_terms: D000241:Adenosine / Q000031:analogs & derivatives / Q000378:metabolism / Q000494:pharmacology; D000248:Adenosine Kinase / Q000037:antagonists & inhibitors / Q000737:chemistry*; D000818:Animals; D018053:Arsenites / Q000494:pharmacology; D001665:Binding Sites; D002847:Chromatography, Agarose; D003958:Diamide / Q000494:pharmacology; D004112:Dimercaprol / Q000494:pharmacology; D004220:Disulfides / Q000737:chemistry; D004791:Enzyme Inhibitors / Q000494:pharmacology; D005033:Ethylmaleimide / Q000494:pharmacology; D007700:Kinetics; D007893:Leishmania donovani / Q000201:enzymology*; D008741:Methyl Methanesulfonate / Q000031:analogs & derivatives / Q000494:pharmacology; D013050:Spectrometry, Fluorescence; D013438:Sulfhydryl Compounds / Q000737:chemistry / Q000378:metabolism*
chemical_list: D018053:Arsenites; D004220:Disulfides; D004791:Enzyme Inhibitors; D013438:Sulfhydryl Compounds; D004112:Dimercaprol; D003958:Diamide; C014674:methyl methanethiosulfonate; D008741:Methyl Methanesulfonate; D000248:Adenosine Kinase; D000241:Adenosine; C015001:arsenite; D005033:Ethylmaleimide