Affinity Labeling of the Active Site of Rabbit Muscle Adenylosuccinate Lyase by 2-[(4-Bromo-2,3-Dioxobutyl)Thio]Adenosine 5‘-Monophosphate

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Abstract
Rabbit muscle adenylosuccinate lyase upon incubation with 7.5−50 μM 2-[(4-bromo-2,3-dioxobutyl)thio]adenosine 5‘-monophosphate (2-BDB-TAMP) in 0.05 M PIPES buffer, pH 7.0 and 10 °C, gives a time dependent biphasic inactivation. The rate of inactivation exhibits a nonlinear dependence on the concentration of 2-BDB-TAMP, which can be described by reversible binding of reagent to the enzyme (KI = 8.5 μM, 5.2 μM) prior to the irreversible reaction, with maximum rate constants of 0.319 and 0.027 min-1 for the fast and slow phases, respectively. The enzyme is a tetramer, with subunits of 50 000 Da. When the enzyme was 90% inactivated, 0.84 mol of reagent/mol of subunit was incorporated as measured by protein-bound phosphate analysis; similar results were obtained using 2-BDB-[14C]TAMP. Complete protection against inactivation and incorporation was afforded by 1 mM 5‘-AMP and by 0.1 mM 5‘-AMP + 5 mM fumarate (the natural products of adenylosuccinate hydrolysis) but not by 0.1 mM 5‘-AMP alone, 5 mM fumarate alone, or 0.1 mM 5‘-AMP + 5 mM maleate or 5 mM succinate. These studies suggest that 2-BDB-TAMP inactivates adenylosuccinate lyase by specific reaction at the substrate binding site, with negative cooperativity between subunits accounting for the appearance of two phases of inactivation. Cleavage of 2-BDB-TAMP-modified enzyme with cyanogen bromide and subsequent separation of peptides by reverse phase HPLC gave only one radioactive peak. This radioactive peptide was further digested with papain and the target site of the 2-BDB-TAMP reaction was identified as Arg112. We conclude that Arg112 is located in the substrate binding site of rabbit muscle adenylosuccinate lyase.