Fluorescence energy transfer between cysteine 199 and cysteine 343: evidence for magnesium ATP-dependent conformational change in the catalytic subunit of cAMP-dependent protein kinase

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Abstract
The actalytic subunit of cAMP-dependent protein kinase has two cysteine residues, Cys 199 and Cys 343, which are protected against alkylation by MgATP [Nelson, N. C., and Taylor, S. S. (1981) J. Biol. Chem. 256, 3743]. While Cys 199 is in close proximity to the active site of the catalytic subunit and is probably directly protected against alkylation by MgATP, the mechanism by which MgATP prevents alkylation of Cys 343 is unclear. To determine whether MgATP directly protects Cys 343 from alkylation by being in close proximity to both Cys 199 and the MgATP binding site, fluorescence resonance energy transfer techniques were used to measure the distance between Cys 199 and Cys 343. Two different donor-acceptor pairs containing 4-[N-[(iodoacetoxy)ehtyl]-N-methylamino]-7-nitrobenz-2-oxa-1,3-diazole at Cys 199 as the acceptor and either 3,6,7-trimethyl-4-(bromomethyl)-1,5-diazabicyclo[3.3.0]octa-3,6-diene-2,8-dione or N-(iodoacetyl)-N''-(5-sulfo-1-naphthyl)ethylenediamine at Cys 343 as the donor were prepared following the method described in the preceding paper [First, E. A., and Tayler, S. S. (1989) Biochemistry (preceding paper in this issue)]. From the efficiencies of fluorescence resonance energy transfer for each donor-acceptor pair, the distance between Cys 199 and Cys 343 was estimated to be between 31 and 52 .ANG.. Since Cys 199 is close to the MgATP binding site and since MgATP cannot extend beyond a distance of 15 .ANG., it is unlikely that Cys 343 at a distance of at least 31 .ANG. from Cys 199 is in direct contact with the bound nucleotide. The 31-52-.ANG. distance between the two cysteines suggests instead that the binding of MgATP induces a conformational change in the catalytic subunit, which leads to the protection of Cys 343.