Affinity labelling of 5'-nucleotidases with 5'-p-fluorosulphonylbenzoyladenosine.
5'-Nucleotidases play an important role in the metabolism of nucleosides; for example, the hydrolysis of AMP generates adenosine, which can modulate a variety of cellular functions. We have used the membrane-bound AMPase from chicken gizzard and a secreted form of these enzymes to analyse their modification by the substrate analogue 5'-p-fluorosulphonylbenzoyladenosine (5'-FSBA). 5'-FSBA irreversibly inactivates 5'-nucleotidases by means of covalent modification of the proteins. ATP, a competitive inhibitor of chicken gizzard and snake-venom 5'-nucleotidase, abolished the inactivation by 5'-FSBA, demonstrating that the inactivation was due to the modification of amino acid residues essential for AMPase activity. We have synthesized radioactive 5'-FSBA, which was employed for the radiolabelling of chicken gizzard 5'-nucleotidase. Incorporation of radioactivity was completely abolished in the presence of ATP, which showed that 5'-FSBA acted by the selective modification of amino acid residues at the active site whereas other potential reactive residues of the protein were not attacked. Limited proteolysis of affinity-labelled chicken gizzard 5'-nucleotidase permitted the identification of digestion products containing the catalytic centre. Pseudo-first-order kinetics indicate that modification of a minimum of one amino acid side chain at the active centre is sufficient to result in inactivation of both chicken gizzard and snake-venom 5'-nucleotidases. Incorporation of the radioactive p-sulphonylbenzoyladenosine moiety parallels the inactivation of 5'-nucleotidase by 5'-FSBA and further substantiated the idea that modification of one amino acid residue at the active centre results in loss of the AMPase activity.
Stochaj
U
U
Institut für Anatomie und Zellbiologie, Marburg, Federal Republic of Germany.
Mannherz
H G
HG
D015720:5'-Nucleotidase / Q000378:metabolism*; D000241:Adenosine / Q000031:analogs & derivatives* / Q000494:pharmacology; D000255:Adenosine Triphosphate / Q000378:metabolism; D000345:Affinity Labels; D000596:Amino Acids / Q000032:analysis; D000818:Animals; D001665:Binding Sites; D002645:Chickens; D005895:Gizzard, Avian / Q000201:enzymology; D006863:Hydrogen-Ion Concentration; D008970:Molecular Weight; D010449:Peptide Mapping
D000345:Affinity Labels; D000596:Amino Acids; C010959:5'-(4-fluorosulfonylbenzoyl)adenosine; D000255:Adenosine Triphosphate; D015720:5'-Nucleotidase; D000241:Adenosine