Mechanistic studies on human liver Biliverdin-IX beta reductase

Abstract

The reaction mechanism of human biliverdin-IXβ reductase (BVR-B) has been investigated using a number of approaches. The preference for NADPH over NADH has been examined using site-directed mutagenesis. The crystal structure of BVR-B, reported by Pereira et al. (2001), shows that Arg-35 and Arg-78 interact with the 2'- phosphate of NADP+ and indicates Arg-39 may also be involved in NADPH binding. BVR-B exhibits pronounced substrate inhibition preventing detailed kinetic characterisation of the enzyme's biliverdin reductase activity. BVR-B has previously been shown to be identical to flavin reductase. The flavin reductase activity of BVR-B was used in an initial rate study that revealed that the dominant residue in determining the enzyme’s preference for NADPH is Arg-35. Replacement of this residue with alanine or serine resulted in the apparent Km NADPH approaching that of the apparent Km for NADH. The k cat, with FMN as the second substrate, was only slightly reduced. Replacing Arg-78 and Arg-39 had essentially no effect on the Km NADPH or the k cat values with FMN as the variable substrate. These results show good agreement with preliminary experiments that model NADP+ desorption using steered molecular dynamics. These calculations, performed by Dr Liam Smith show that release of the guanido-terminus of Arg-35 from its "locking" position over the adenine of NADP+ appears to permit the "unwrapping" o f Arg-78 and Arg-39.