Complexation of Membrane-Bound Enzyme Systems

Abstract

Cytochrome P450, NADPH-Cytochrome P450 Reductase, Membrane-Binding DomainsThe effect of changes in the N-terminal membrane-binding domain of cytochrome P450 forms and NADPH-cytochrome P450 reductase types on the cytochrome P450-dependent monooxygenase activities, has been examined. The nifedipine oxidase activity of two human P450 forms (CYP3A4, CYP3A4NF14) which differ only in their primary structure by ten amino acid residues in the N-terminal membrane-binding domain, yields nearly the same catalytic cycle time x =2.65 ± 0.15 s, due to their identical cytosolic catalytic protein structure. In contrast, the complex formation process ([P450]+ [reductase] [complex]) described by the dissociation constant K D at high substrate concentration ([5]»/C5) and low product con centration ([ P ] « /^) is determined to be /CD/[P450]o = 0.3 and 2.0, respectively. These values reflect large differences in the affinity of both P450 forms for the same type of reductase which is only due to their modified membrane-binding domains. In the present work, it has been shown for the first time, that the membrane-binding domain of cytochrome P450 en zymes determines the complexation process of the binary P450:reductase system. Further more, the nifedipine oxidase activity of the human CYP3A4 form reconstituted with two different types of reductase from human and rabbit also has the same catalytic cycle time x = 2.65 ± 0.15 s. This result is based on the similarity of the primary structure of the cytosolic catalytic domain of both reductase types. However, the complex was formed with different dissociation constants of A T D/[P450]o = 0.3 and 4.7, respectively. This different affinity of both reductase types to the same P450 form is interpreted as a consequence of the substantial alteration of the amino acids in the N-terminal primary structure of their membrane-binding domains. 7-Ethoxycoumarin O-deethylase activity of two rat P450 forms (CYP2B1 and CYP1A1) were reconstituted with the same rat reductase. The catalytic cycle time for each P450 form is x = 1.8 and 0.6 s, respectively. Correspondingly, the complex formation process controlled by the dissociation constant K n has changed from /CD/[P450]o = 2.3 to 1.7, respec tively. This is because both forms differ in their cytosolic as well as in their membranebinding domains.

Drug-Drug Interactions Evaluated by a Highly Active Reconstituted Native Human Cytochrome P4503A4 and Human NADPH-Cytochrome P450 Reductase System

“…We were able to show that the complex formation process ( higher for the shortened CYP3A4 protein than for the native full-length CYP3A4 enzyme [12,13]. This means that the modification of the N-terminal amino acid sequence changes the catalytic properties by this factor.…”

Aromatase and breast cancer: W39R, an inactive protein

“…For instance, Lee-Robichaud et al (34) have described the importance of arginine residues which are involved in protein -protein interaction between cytochrome b5 and human CYP17 and consequently affect the lyase activity of the enzyme required for androgen formation. Moreover, Muller-Enoch & Gruler (35) have recently reported that the N-terminal binding domain of cytochrome P450 enzymes determines the complexation process of the binary P450 reductase system. We also hypothesized that R39 may form a salt-bridge with another glutamic acid residue located far away in the sequence, as has been described in CYP19 between R9 and E296 (36), and consequently could prevent the interaction between P450 aromatase and NADPH reductase.…”

Fluorescence Resonance Energy Transfer Analysis of Cytochromes P450 2C2 and 2E1 Molecular Interactions in Living Cells

“…The decreased efficiency of FRET for OEC compared with full-length P450 2C2 suggests that the SA may also contribute to the interaction. In this regard, differences in the SA of P450s have been shown to affect the affinity to P450 reductase (41).…”