The ATP/Mg²⁺ Balance Affects the Degradation of Short Fluorogenic Substrates by the 20S Proteasome

Proteasomes hydrolyze most cellular proteins. The standard reaction to determine proteasome activity in cellular lysate or elsewhere contains AMC-conjugated peptide substrate, ATP, Mg²⁺, and DTT. ATP and Mg²⁺ are included to maintain 26S proteasome functionality. However, most cellular proteasomes are 20S proteasomes, and the effects of ATP on the turnover of fluorogenic substrates by 20S complexes are largely unknown. Here, we evaluated the effect of ATP alone or in combination with Mg²⁺ on the degradation of AMC-conjugated fluorogenic substrates by purified 20S proteasomes. Degradation of substrates used to determine chymotrypsin-, caspase- and trypsin-like proteasome activities was gradually decreased with the rise of ATP concentration from 0.25 to 10 mM. These effects were not associated with the blockage of the proteasome catalytic subunit active sites or unspecific alterations of AMC fluorescence by the ATP. However, ATP-induced peptide degradation slowdown was rescued by the addition of Mg²⁺. Moreover, the substrate degradation efficacy was proportional to the Mg²⁺/ATP ratio, being equal to control values when equimolar concentrations of the molecules were used. The obtained results indicate that when proteasome activity is assessed, the reciprocal effects of ATP and Mg²⁺ on the hydrolysis of AMC-conjugated fluorogenic substrates by the 20S proteasomes should be considered.