Affinity Labeling of the active site of rabbit muscle adenylosuccinate lyase by 2-[(4-bromo-2.3-dioxobutyl)thio] adenosine 5'-monophosphate

Biochemistry
S U Gite, Roberta F Colman

Abstract

Rabbit muscle adenylosuccinate lyase upon incubation with 7.5-50 muM 2 -[(4-bromo-2.3-dioxobutyl)thio]adenosine 5'-monophosphate (2-BDB-TAMP) in 0.05 M PIPES buffer, ph 7.0 and 10 degrees C, gives a time dependent biphasic inactivation. The rate of inactivation exhibits a nonlinear dependence on the concentration 2-BDB-TAMP, which can be described by reversible binding of reagent to the enzyme (K1=8.5 microM. 5.2 microM) 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 s...Continue Reading

References

Nov 15, 1979·Analytical Biochemistry·P A LanzettaO A Candia
Jan 1, 1992·Journal of Inherited Metabolic Disease·J JaekenG Van den Berghe
Nov 9, 1989·Biochimica Et Biophysica Acta·B A BarshopH E Gruber
Jan 1, 1983·Annual Review of Biochemistry·R F Colman

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