Probing the function(s) of active-site arginine residue in Leishmania donovani adenosine kinase.
The presence of arginine at the active site of Leishmania donovani adenosine kinase was studied by chemical modification, followed by the characterization of the modified enzyme. The arginine-specific reagents phenylglyoxal (PGO), butane-2,3-dione and cyclohexane-1,2-dione all irreversibly inactivated the enzyme. In contrast, adenosine kinase from hamster liver was insensitive to these reagents. The inactivation of the enzyme by PGO followed pseudo-first-order kinetics, with a second-order rate constant of 39.2 min-1.M-1. Correlation between the stoichiometry of PGO modification and extent of inactivation indicated that modification of a single residue per molecule suffices for the loss of activity. Reactivity of the essential arginine residue towards PGO was affected by the presence of adenosine (Ado) and other competing alternative substrates, consistent with an arginine residue located proximal to the Ado-binding site. The enzyme showed an intrinsic fluorescence with an emission maximum at 340 nm when excited at 295 nm. The protein fluorescence was partially quenched on addition of Ado. PGO modification also led to significant quenching of the fluorescence. However, the fluorescence of the Ado-protected enzyme, which displayed 82% of the original activity after PGO treatment, was retained. The kinetic analyses of the partially modified enzyme showed an increase in the Km for Ado from 14 to 55 microM. Furthermore, the inability of the modified enzyme to bind to 5'-AMP-Sepharose 4B affinity column provided additional evidence that modification is attended by decrease in affinity of the enzyme for Ado. The results are consistent with the interpretation that modification of the active-site arginine residue affects activity by interfering with the binding of the substrate to the active site.
Ghosh
M
M
Leishmania Group, Indian Institute of Chemical Biology, Jadavpur, Calcutta.
Datta
A K
AK
D000241:Adenosine / Q000378:metabolism; D000248:Adenosine Kinase / Q000037:antagonists & inhibitors / Q000737:chemistry / Q000378:metabolism*; D000818:Animals; D001120:Arginine / Q000378:metabolism*; D001665:Binding Sites; D002852:Chromatography, Ion Exchange; D006224:Cricetinae; D003512:Cyclohexanones / Q000494:pharmacology; D004852:Epoxy Compounds / Q000494:pharmacology; D007700:Kinetics; D007893:Leishmania donovani / Q000201:enzymology*; D008099:Liver / Q000201:enzymology; D010658:Phenylglyoxal / Q000494:pharmacology; D013050:Spectrometry, Fluorescence
D003512:Cyclohexanones; D004852:Epoxy Compounds; C007366:diepoxybutane; C018031:1,2-cyclohexanedione; D001120:Arginine; D000248:Adenosine Kinase; D000241:Adenosine; D010658:Phenylglyoxal