Locus of the catalytic sites of udp-glucose dehydrogenase in the native enzyme hexamer as delineated by fluorescence energy transfer

UDP-glucose dehydrogenase is comprised of six identical subunits arranged in a hexagonal manner. The half-sites reactivity behavior of the enzyme suggests that the array has 32 symmetry, i.e., it is a trimer of dimers. Since the catalytic site thiol groups are biphasically alkylated, one can prepare enzyme which has three catalytic sites covalently blocked with a fluorescent donor and three with an acceptor. Peptide mapping of tryptic digests of enzyme so modified shows that < 5% of the fluorophores are on noncatalytic site peptides. The AEDANS group has been used as a donor with either fluorescein, eosin, or nitrobenzoxadiazole as the acceptor. The extent of incorporation was determined from the radioactivity and/or optical absorbance of the modified protein. Energy transfer efficiencies were evaluated routinely from the size of donor fluorescence quenching, after it was certain that sensitized fluorescence data gave comparable results.