Adenosine triphosphate-dependent degradation of a fluorescent lambda N substrate mimic by lon protease

Escherichia coli Lon exhibits a varying degree of energy requirement toward hydrolysis of different substrates. Efficient degradation of protein subst rates requires the binding and hydrolysis of ATP such that the intrinsic AT Pase of Lon is enhanced during protein degradation. Degradation of syntheti c tetrapeptides, by contrast, is achieved solely by ATP binding with concom itant inhibition of the ATPase activity. In this study, a synthetic peptide (FRETN 89-98), containing residues 89-98 of lambda N protein and a fluores cence donor (anthranilamide) and quencher (3-nitrotyrosine), has been exami ned for ATP-dependent degradation by E, coli and human Lon proteases, The c leavage profile of FRETN 89-98 by E, coli Lon resembles that of h N degrada tion. Both the peptide and protein substrates are specifically cleaved betw een Cys93 and Ser94 with concomitant stimulation of Lon's ATPase activity. Furthermore, the degradation of FRETN 89-98 is supported by ATP and AMPPNP but not ATP gammaS nor AMPPCP, FRETN 89-98 hydrolysis is eight times more e fficient in the presence of 0.5 mM ATP compared to 0.5 mM AMPPNP at 86 muM peptide. The ATP-dependent hydrolysis of FRETN 89-98 displays sigmodial kin etics, The k(cat), [S](0.5), and the Hill coefficient of FRETN 89-98 degrad ation are 3.2 +/- 0.3 s(-1), 106 +/- 21 muM and 1.6 respectively. (C) 2001 Academic Press.