Affinity modification of NADPH-cytochrome P-450 reductase

Abstract

The active centre of NADPH-cytochrome P-450 reductase contains the lysine residue essential for catalytic activity. Chemical modification of ε-amino group of this lysine residue is the subject of the present study. To modify the ε-amino group we have employed the periodate-oxidized NADP+ and NAD+ (o-NAD(P)). The both reagents have appeared to be the competitive inhibitors of NADPH-cytochrome P-450 reductase (Ki for o-NADP ∼ 10 μM, Ki for o-NAD > 100 μM). However, o-NADP has not a covalency bond with reductase, whilst o-NAD modifies the reductase at the binding site of NADPH. A protective effect of NADP+ and the labeling extent close to unity (0.7) at deep reductase inactivation indicate the affinity type of this modification. Different results of reductase modification by either o-NADP or o-NAD may be due to the difference in the structures of the analogs bound to the enzyme active site. © 1988 Academic Press, Inc.