Effects of the inhibitors azide, dicyclohexylcarbodiimide, and aurovertin on nucleotide binding to the three F-1-ATPase catalytic sites measured using specific tryptophan probes

Equilibrium nucleotide binding to the three catalytic sites of Escherichia coli F-1-ATPase was measured in the presence of the inhibitors azide, dicyc lohexylcarbodiimide, and aurovertin to elucidate mechanisms of inhibition. Fluorescence signals of beta-Trp-331 and beta-Trp-148 substituted in cataly tic sites were used to determine nucleotide binding parameters. Azide broug ht about small decreases in K-d(MgATP) and K-d(MgADP), Notably, under MgATP hydrolysis conditions, it caused all enzyme molecules to assume a state wi th three catalytic site-bound MgATP and zero bound MgADP, These results rul e out the idea that azide inhibits by "trapping" MgADP, Rather, azide block s the step at which signal transmission between catalytic sites promotes mu ltisite hydrolysis, Aurovertin bound with stoichiometry of 1.8 (mol/mol of F-1) and allowed significant residual turnover. Cycling of the aurovertin-f ree beta-subunit catalytic site through three normal conformations was indi cated by MgATP binding data. Aurovertin did not change the normal ratio of 1 bound MgATP/2 bound MgADP in catalytic sites. The results indicate that i t acts to slow the switch of catalytic site affinities ("binding change ste p") subsequent to MgATP hydrolysis, Dicyclohexylcarbodiimide shifted the ra tio of catalytic site-bound MgATP/MgADP from 1:2 to 1.6:1.4, without affect ing K-d(MgATP) values. Like azide, it also appears to affect activity at th e step after MgATP binding, in which signal transmission between catalytic sites promotes MgATP hydrolysis.