The cytochrome P450 responsible for the debrisoquine/sparteine polymorphism (P450 2D6) has been produced in large quantities by expression of a modified cDNA in baculovirus. A polyhistidine extension was incorporated at the C-terminus of the expressed protein, which, after purification of the protein on a nickel-agarose column, could be removed proteolytically by treatment with thrombin. Purified yields of P450 2D6 were 2.4 mg from 700 mL of cell culture. The protein had a greater than 90% heme content and was fully active, having no residual absorbance at 420 nm in the reduced CO complex. The quantities produced allowed direct study of the interaction of the substrate codeine with the enzyme by paramagnetic relaxation effects on the NMR spectrum of the substrate. Distances between the heme iron atom and substrate protons were calculated from these experiments, and the orientation of the substrate in the binding pocket was determined. This showed that codeine was bound with the methoxy group of the molecule closest to the heme iron (iron-methyl proton distance of 3.1 +/- 0.1 A), consistent with the observed O-demethylation to morphine. A model of the complex Of P450 2D6 with codeine was built from a multiple sequence and structure alignment of the known crystal structures for P450s, incorporating the experimental constraints derived from the NMR studies. This showed that the overall fold Of P450 2D6 is more similar to that of P450 BM3 than to either P450 cam or P450 terp. Codeine binds to P450 2D6 so that the methoxy group is directly above the A ring of the heme, while the basic nitrogen interacts with the carboxylate of aspartate 301.

The binding of the substrates sodium laurate and sodium 12-bromolaurate to the heme-containing domain of Bacillus megaterium cytochrome P450 BM3 (CYP102) has been studied by measurement of the relaxation effects of the unpaired electrons of the heme iron on the protons of water and of the bound substrates. Substrate binding leads to a conversion of the heme iron from a low-spin to a high-spin state, as shown by changes in the optical spectrum. The relaxation measurements show that this is accompanied by expulsion of water from the sixth coordination position of the iron, the distance between the iron and the water protons increasing from 2.6 to 5.2 A. Corresponding relaxation measurements on the substrate protons lead to the determination of a number of distances between the iron and protons of the bound substrate and, hence, to information on the position and orientation of the substrate in the binding site. Laurate and 12-bromolaurate are found to bind in a very similar way, in an extended conformation with the carboxylate probably close to Arg47 and the other end of the chain 7.6-7.8 A from the heme iron. It is shown that laurate and pyridine can bind simultaneously to the P450 domain and that the iron-laurate distances in this ternary complex are not significantly different from those in the binary complex. These observations are compared with those on the substrate complex of cytochrome P450 cam, and their implications for structural changes involved in the catalytic cycle are discussed.

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13C NMR is used to detect ionizations within a trypsin-chloromethyl ketone inhibitor complex. The pKa values observed are compared with those predicted by free-energy relationships. For the denatured/autolyzed inhibitor complex, a pKa = 5.26 is observed, which is assigned to the ionization of the imidazole of histidine-57. For the intact inhibitor complex a pKa = 7.88 is determined. This pKa is assigned to the ionization of the hemiketal hydroxyl (pKa = 7.88-8.1) and provides the first direct evidence that the serine proteases are able to stabilize the oxyanion of tetrahedral adducts. Indirect evidence is adduced that the imidazole pK1 of histidine-57 is greater than or equal to 8.1. Line-broadening studies suggest that there may be extra fast exchange line broadening, which could result from rapid tautomeric exchange between neutral and zwitterionic species within the inhibitor complex. The significance of these results for the catalytic mechanism of serine proteases is discussed.

Phenylalanine 87 of Bacillus megaterium cytochrome P450 BM3, a residue close to the heme in the substrate binding pocket, has been replaced by alanine by site-directed mutagenesis. The substitution had no effect on the rate of hydroxylation of laurate and increased the affinity for laurate of both the intact enzyme and its heme domain by 2.6-6-fold in the ferric state. NMR paramagnetic relaxation measurements showed that in the initial ferric enzyme-substrate complex, where the substrate binds relatively far from the heme, the substitution had no effect on the position or orientation of the bound substrate. However, in the next intermediate in the catalytic cycle, the reduced enzyme, the position of the bound substrate was altered so that the terminal methyl group was 3.1 A from the iron in the mutant, compared to 5.1 A in the wild-type enzyme. Analysis of the products of the action of the enzyme on laurate and myristate showed that the mutant catalyzed hydroxylation almost exclusively at the omega position, in marked contrast to the wild-type enzyme, with which no hydroxylation at this position was observed.

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1-Methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP), a neurotoxin that produces Parkinsonism symptoms in man, has been examined as a substrate of recombinant human cytochrome P450 2D6. When cumene hydroperoxide is used as an oxygen and electron donor, a single product is formed, identified as 4-phenyl-1,2,3,6-tetrahydropyridine. The K(m) for formation of this product (130 microM) is in agreement with the dissociation constants for MPTP binding to the enzyme determined by optical and nuclear magnetic resonance (NMR) spectroscopy. When the reaction is carried out with nicotinamide adenine dinucleotide phosphate (reduced) (NADPH) and recombinant human NADPH-cytochrome P450 reductase, a second product, identified as 1-methyl-4-(4'-hydroxyphenyl)-1,2,3,6-tetrahydropyridine, is formed in addition to 4-phenyl-1,2,3,6-tetrahydropyridine. The K(m) values for formation of these two products are 19 microM and 120 microM, respectively. Paramagnetic relaxation experiments have been used to measure distances between the protons of bound MPTP and the heme iron, and these have been used to construct models for the position and orientation of MPTP in the active site. For the cytochrome alone, a single mode of binding was observed, with the N-methyl close to the heme iron in a position appropriate for the observed N-demethylation reaction. In the presence of the reductase, the data were not consistent with a single mode of binding but could be explained by the existence of two alternative orientations of MPTP in the active site. One of these, characterized by a dissociation constant of 150 microM, is essentially identical to that observed in the absence of the reductase. In the second, which has a K(d) of 25 microM, the MPTP is oriented so that the aromatic ring is close to the heme iron, in a position appropriate for p-hydroxylation leading to the formation of the product seen only in the presence of the reductase. In the case of codeine, another substrate for cytochrome P450 2D6, the addition of reductase had no effect on the nature of the product formed, the dissociation constant, or the orientation in the binding site. These observations show that NADPH-cytochrome P450 reductase has an allosteric effect on the active site of cytochrome P450 2D6 that affects the binding of some substrates but not others.