1-METHYL-4-PHENYL-1,2,3,6-TETRAHYDROPYRIDINE AS A SUBSTRATE OF CYTOCHROME-P450 2D6 - ALLOSTERIC EFFECTS OF NADPH-CYTOCHROME P450 REDUCTASE

1-Methyl-4-phenyl-1,2,3,6-tetrahydropyrine (MPTP), a neurotoxin that p roduces Parkinsonism symptoms in man, has been examined as a substrate of recombinant human cytochrome P450 2D6. When cumene hydroperoxide i s used as an oxygen and electron donor, a single product is formed, id entified as 4-phenyl-1,2,3,6-tetrahydropyridine. The K-m for formation of this product (130 mu M) is in agreement with the dissociation cons tants for MPTP binding to the enzyme determined by optical and nuclear magnetic resonance (NMR) spectroscopy. When the reaction is carried o ut with nicotinamide adenine dinucleotide phosphate (reduced) (NADPH) and recombinant human NADPH-cytochrome P450 reductase, a second produc t, identified as l-4-(4'-hydroxyphenyl)-1,2,3,6-tetrahydropyridine, is formed in addition to LF-phenyl-1,2,3,6-tetrahydropyridine. The K-m v alues for formation of these two products are 19 mu M and 120 mu M, re spectively. Paramagnetic relaxation experiments have been used to meas ure distances between the protons of bound MPTP and the heme iron, and these have been used to construct models for the position and orienta tion of MPTP in the active site. For the cytochrome alone, a single mo de of binding was observed, with the N-methyl close to the heme iron i n a position appropriate for the observed N-demethylation reaction. In the presence of the reductase, the data were not consistent with a si ngle mode of binding but could be explained by the existence of two al ternative orientations of MPTP in the active site. One of these, chara cterized by a dissociation constant of 150 mu M, is essentially identi cal to that observed in the absence of the reductase. In the second, w hich has a K-d of 25 mu M, the MPTP is oriented so that the aromatic r ing is close to the heme iron, in a position appropriate for p-hydroxy lation leading to the formation of the product seen only in the presen ce of the reductase. In the case of codeine, another substrate for cyt ochrome P450 2D6, the addition of reductase had no effect on the natur e of the product formed, the dissociation constant, or the orientation in the binding site. These observations show that NADPH-cytochrome P4 50 reductase has an allosteric effect on the active site of cytochrome P450 2D6 that affects the binding of some substrates but not others.