Properties of a cobalt-reactivated form of yeast alcohol dehydrogenase

Yeast alcohol dehydrogenase (Y-ADH) is a widely studied metal-enzyme for it s well-known biotechnological importance. Although its structure has been e xtensively investigated, some topics still remain controversial (zinc conte nt and role), and various attempts aiming at modifying its structure to imp rove its catalytic properties have been made. In this paper, a metal-substi tuted Y-ADH has been prepared in vitro, in which one Zn atom per molecule ( only one of those directly involved in catalysis) has been substituted by o ne Co atom. The substitution was obtained through zinc removal by a chelati ng treatment (with Chelex 100) followed by cobalt insertion. The zinc conte nt in the native enzyme was preliminarily evaluated (taking care to avoid c ontamination) to be 4.1 +/- 0.1 g-at./molecule. After cobalt substitution, the ratio Zn:Co in the enzyme results to be 3:1. The active Co-Y-ADH has be en compared with the native enzyme: it has lower specific activity (about 5 0%) and lower substrate affinity but greater thermo-resistance and a pH sta bility in a wider range than the native Y-ADH. A similar behavior, as far a s cobalt content, thermo-resistance and pH stability are concerned, but gre ater specific activity and substrate affinity, were shown by an in vivo-sub stituted Co-Y-ADH obtained in a previous study. (C) 2000 Elsevier Science B .V. All rights reserved.