Fluorescence studies of ATP-diphosphohydrolase from Solanum tuberosum var.Desiree

Chemical modification of potato apyrase suggests that tryptophan residues a re close to the nucleotide binding site. K-d values (+/- Ca2+) for the comp lexes of apyrase with the non-hydrolysable phosphonate adenine nucleotide a nalogues, adenosine 5'-(beta,gamma- methylene) triphosphate and adenosine 5 '-(alpha,beta-methylene) diphosphate, were obtained from quenching of the i ntrinsic enzyme fluorescence. Other fluorescent nucleotide analogues (2'(3' )-O-(2,4,6-trinitrophenyl) adenosine 5'-triphosphate, 2'(3')-O-(2,4,6-trini trophenyl) adenosine 5'-diphosphate, 1,N-6-ethenoadenosine triphosphate and 1,N-6-ethenoadenosine diphosphate) were hydrolysed by apyrase in the prese nce of Ca2+, indicating binding to the active site. The dissociation consta nts for the binding of these analogues were calculated from both the decrea se of the protein (tryptophan) fluorescence and enhancement of the nucleoti de fluorescence. Using the sensitised acceptor (nucleotide analogue) fluore scence method, energy transfer was observed between enzyme tryptophans and ethene-derivatives. These results support the view that tryptophan residues are present in the nucleotide-binding region of the protein, appropriately oriented to allow the energy transfer process to occur. (C) 2000 Elsevier Science Ltd. All rights reserved.