SUBSTRATE-BINDING CAUSES MOVEMENT IN THE ATP BINDING DOMAIN OF ESCHERICHIA-COLI ADENYLATE KINASE

Crystallographic evidence suggests that there is a large hinged domain motion associated with substrate binding in adenylate kinase. To test this hypothesis, resonance energy transfer measurements of substrate binding were initiated. Adenylate kinase from Escherichia coli consist s of three domains: the main body of the enzyme with alpha-helical and beta-sheet secondary structure, and domains that close over the AMP a nd ATP binding sites. Four single tryptophan mutants were constructed to map distances. Two tryptophan mutants were positioned at residues 1 33 (Y133W) and 137 (F137W), which are in the domain that closes over t he ATP binding site. Mutant F86W that is located at the AMP binding si te, and mutant S41W that is in the loop that close over AMP, complete the mapping library. Energy transfer was measured between each of thes e tryptophans and [2-(acetylamino)ethyl]amino]naphthalene-1-sulfonic a cid (AEDANS) covalently bound to the single cysteine residue at positi on 77, which is located in the main body of adenylate kinase. The dist ance between the tryptophan of the F137W mutant adenylate kinase and t he AEDANS-labeled Cys-77 decreased by 12.1 Angstrom upon the binding o f the bisubstrate inhibitor P-1,P-5-bis(5'-adenosyl) pentaphosphate (A P(5)A). There were only small alterations in the tryptophan to Cys-77- AEDANS distances in the Y133W, F86W, and S41W mutants upon the binding of AP(5)A, ATP, or AMP, implying that movement of residues 133, 86, a nd 41 in relation to the Cys-77 residue was minimal. These results sug gest that there is significant closure of the ATP binding domain upon the binding of ATP or AP(5)A. Unexpectedly, exposure of the enzyme to AMP also introduced a partial closure of the ATP hinged domain.