PubMed1992
The topological location of the nucleotide substrate binding environments on adenylate kinase has been explored with the fluorescent molecule [4-benzoyl]benzoyl-1-amidofluorescein (BzAF) and the nucleotide analog 3'-O-[4-benzoyl]benzoyl-ATP (BzATP), which are site-directed photoaffinity probes that bind covalently at the individual nucleotide sites. The MgBzATP substituted for MgATP as a substrate for this enzyme, whereas BzAF, which is neither a substrate nor a nucleotide, behaved as a competitive inhibitor for each nucleotide site independently. BzAF could be directed specifically to either of the nucleotide sites by near saturation of the second site with its natural nucleotide substrate. Using this second site blocking approach, each nucleotide site, in turn, could be protected competitively from BzAF-induced photoinhibition by the presence of its natural substrate. This strategy showed that under photolysis, the Kd(BzAF) of 0.1 mM (for the MgATP site) and 0.2 mM (for the AMP site) were nearly identical with the Km(app) values determined for MgATP and AMP, respectively. Pseudo first-order photolysis kinetics with [3H]BzAF revealed covalent binding stoichiometries for full inhibition of 1 mol of probe/mol of enzyme at either site. Thus, we prepared the binary complex ([3H]BzAF-enzyme) by photo-labeling the MgATP site-blocked enzyme with [3H]BzAf to 1:1 molar stoichiometry. Tryptic digestion followed by partial sequencing of the [3H]BzAF-labeled enzyme disclosed that BzAF was bound specifically within the peptide span Gly64-->Arg77. This amino acid domain therefore probably constitutes the neighborhood identifiable with AMP binding. Furthermore, we also achieved double photocovalent labeling of adenylate kinase with both photoprobes, by first cross-linking with approximately 1 mol of MgBzATP, followed by approximately 0.9 mol of [3H]BzAF, thus generating a ternary covalent complex. Comparison of the fluorescence of the binary species and the ternary (BzATP-enzyme-[3H]BzAF) complex revealed altered fluorescence emission profiles, which could indicate that a conformational change occurs during formation of the ternary (or transition state) complex.