Active site thiol(s) in Leishmania donovani adenosine kinase: comparison with hamster enzyme and evidence for the absence of regulatory adenosine binding site

Abstract

Adenosine kinase (ATP, adenosine 5'-phosphotransferase, E.C. 2.7.1.20) from Leishmania donovani, unlike adenosine kinase from other known eukaryotic sources, does not elicit an inhibitory response at high concentrations of adenosine. The mechanistic basis for this unique catalytic behavior of the parasite enzyme has been probed with the help of chemical modification and enzyme inhibition kinetics experiments. The use of cysteine-directed reagents has shown that chemical integrity of cysteinyl residues is essential for the expression of functional activity of the enzyme. Thiol group titration revealed that the enzyme contains 3 cysteine residues. However, in contrast to adenosine kinase from other sources, inactivation of the parasite enzyme could be correlated with alkylation of 2 cysteinyl residues. Adenosine, but not ATP, protected 2 thiols against -SH blocker-mediated inactivation of the enzyme. The thiol groups were shown to map at positions corresponding to approximately 16, 22, and 36 kDa sites from the protein's N-terminal end. The functions of 2 thiols at the catalytic site were analyzed. Quantitative evaluation of the protection of the enzyme conferred by adenosine against NEM-mediated blockage of functional thiol groups yielded a 'protection constant' (K_p^Ad) of 3.4 μ M, while the dissociation constant (K_s^Ad) of the enzyme-substrate complex was 2.7 μ M, hence supporting involvement of the same site in both processes, namely catalysis and protection. The overall results were therefore interpreted as showing that (a) the leishmanial enzyme, in contrast to adenosine kinase from other sources, contains 2 functional thiol groups at the catalytic site; and (b) the enzyme binds adenosine exclusively through the catalytic site and as a consequence is not amenable to inhibition at high adenosine concentrations. The biological implications of these observations in leishmanial parasites, which lack the ability to synthesize purines de novo, are discussed.