The kinetic mechanism and metal content of Haloferax mediterranei NAD(P)(+)-glucose dehydrogenase have been investigated. The kinetic mechanism has been determined by initial rate and inhibition studies. Initial velocity studies 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 patterns agree with an ordered binding of NADP(+) and D-glucose, followed by an ordered release of gluconolactone and NADPH. The activity of Hf: mediterranei glucose dehydrogenase was markedly dependent on the concentration of metal ions. Inactivation by metal chelators and reactivation by certain divalent ions indicated that glucose dehydrogenase from Hf. mediterranei contains 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 protein, which corresponds to the binding of two atoms of Zn(II) per subunit. Alignment of the N-terminal sequence of glucose dehydrogenase from Hf. mediterranei with medium chain zinc-containing dehydrogenases reveals a clear similarity between them, suggesting that glucose dehydrogenase from Hf. mediterranei belongs to this family. (C) 2000 Elsevier Science B.V. All rights reserved.
|Number of pages||9|
|Journal||Journal of Molecular Catalysis B: Enzymatic|
|Publication status||Published - 2000|