NAD(P)(+)-glucose dehydrogenase from Haloferax mediterranei: kinetic mechanism and metal content

The kinetic mechanism and metal content of Haloferax mediterranei NAD(P)(+) -glucose dehydrogenase have been investigated. The kinetic mechanism has be en determined by initial rate and inhibition studies. Initial velocity stud ies were performed with D-glucose as well as with the alternative substrate D-xylose, with NADP(+) as coenzyme. The results show that the mechanism is sequential with respect to substrate addition. The product inhibition patt erns agree with an ordered binding of NADP(+) and D-glucose, followed by an ordered release of gluconolactone and NADPH. The activity of Hf: mediterra nei glucose dehydrogenase was markedly dependent on the concentration of me tal ions. Inactivation by metal chelators and reactivation by certain dival ent ions indicated that glucose dehydrogenase from Hf. mediterranei contain s tightly bound metal ions which are essential for activity. Metal analyses demonstrated that the enzyme binds 3.6 +/- 0.3 mol of Zn(II)/mol of protei n, which corresponds to the binding of two atoms of Zn(II) per subunit. Ali gnment of the N-terminal sequence of glucose dehydrogenase from Hf. mediter ranei with medium chain zinc-containing dehydrogenases reveals a clear simi larity between them, suggesting that glucose dehydrogenase from Hf. mediter ranei belongs to this family. (C) 2000 Elsevier Science B.V. All rights res erved.