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.

1. We describe the effects on channel function of changing an aspartate residue (Asp172) in a membrane‐spanning alpha‐helix of the murine inward rectifier, IRK1, by site‐directed mutagenesis. 2. Alteration of Asp172 to Glu (charged) or to Gln or Asn (polar but uncharged) produced functional channels showing inward rectification, though rectification was weaker with Gln and Asn. 3. Intrinsic gating around the potassium equilibrium potential, EK, was conserved only if the charge on residue 172 was conserved. Currents through channels with Gln or Asn in this position showed no time dependence under hyperpolarization. 4. The change from Asp to Gln also reduced the affinity for internal Mg2+ at least fivefold, indicating that Asp172 also forms part of the site for Mg2+ blockage. 5. The consequences for channel structure of Asp172 lining the pore are discussed.

Structural models have been produced for three types of non-NMDA inotropic glutamate receptors: an AMPA receptor, GluR1, a kainate receptor, GluR6; and a low-molecular-weight kainate receptor from goldfish, GFKAR alpha. Modeling was restricted to the domains of the proteins that bind the neurotransmitter glutamate and that form the ion channel. Model building combined homology modeling, distance geometry, molecular mechanics, interactive modeling, and known constraints. The models indicate new potential interactions in the extracellular domain between protein and agonists, and suggest that the transition from the "closed" to the "open" state involves the movement of a conserved positive residue away from, and two conserved negative residues into, the extracellular entrance to the pore upon binding. As a first approximation, the ion channel domain was modeled with a structure comprising a central antiparallel beta-barrel that partially crosses the membrane, and against which alpha-helices from each subunit are packed; a third alpha-helix packs against these two helices in each subunit. Much, but not all, of the available data were consistent with this structure. Modifying the beta-barrel to a loop-like topology produced a model consistent with available data.

Cytochrome P-450 CYP2D6, human debrisoquine hydroxylase, metabolizes more than 30 prescribed drugs, the vast majority of which are small molecules containing a basic nitrogen atom. In contrast, the similar mouse protein Cyp2d-9 was first characterized as a testosterone 16alpha-hydroxylase. No common substrates have been reported for the two enzymes. Here we investigate the structural basis of this difference in substrate specificity. We have earlier used a combination of NMR data and homology modelling to generate a three-dimensional model of CYP2D6 [Modi, Paine, Sutcliffe, Lian, Primrose, Wolf, C.R. and Roberts (1996) Biochemistry 35, 4541-4550]. We have now generated a homology model of Cyp2d-9 and compared the two models to identify specific amino acid residues that we believe form the substrate-binding site in each protein and therefore influence catalytic selectivity. Although there are many similarities in active site structure, the most notable difference is a phenylalanine residue (Phe-483) in CYP2D6, which in the model is located such that the bulky phenyl ring is positioned across the channel mouth, thus limiting the size of substrate that can access the active site. In Cyp2d-9, the corresponding position is occupied by an isoleucine residue, which imposes fewer steric restraints on the size of substrate that can access the active site. To investigate whether the amino acid residue at this position does indeed influence the catalytic selectivity of these enzymes, site-directed mutagenesis was used to change Phe-483 in CYP2D6 to isoleucine and also to tryptophan. CYP2D6, Cyp2d-9 and both mutant CYP2D6 proteins were co-expressed with NADPH cytochrome P-450 reductase as a functional mono-oxygenase system in Escherichia coli and their relative catalytic activities towards bufuralol and testosterone were determined. All four proteins exhibited catalytic activity towards bufuralol but only Cyp2d-9 catalysed the formation of 16alpha-hydroxytesterone. Uniquely, the CYP2D6F483I mutant acquired the ability to metabolize testosterone to a novel product, which was identified by MS and proton NMR spectroscopy as 15alpha-hydroxytestosterone. NMR spin relaxation experiments were used to measure distances between the haem iron and protons of testosterone bound to the CYP2D6F483I mutant. These experiments demonstrate that very minor modifications to the active site structure of CYP2D6 can have a profound influence on the substrate specificity of the enzyme.

Kir2.1 channels are blocked by Rb+ and Cs+ in a voltage‐dependent manner, characteristic of many inward rectifier K+ channels. Mutation of Ser165 in the transmembrane domain M2 to Leu (S165L) abolished Rb+ blockage and lowered Cs+ blocking affinity. At negative voltages Rb+ carried large inward currents. A model of the Kir2.1 channel, built by homology with the structure of the Streptomyces lividans K+ channel KcsA, suggested the existence of an intersubunit hydrogen bond between Ser165 and Thr141 in the channel pore‐forming P‐region that helps stabilise the structure of this region. However, mutations of Thr141 and Ser165 did not produce effects consistent with a hydrogen bond between these residues being essential for blockage. An alternative alignment between the M2 regions of Kir2.1 and KcsA suggested that Ser165 is itself a pore‐lining residue, more directly affecting blockage. We were able to replace Ser165 with a variety of polar and non‐polar residues, consistent with this residue being pore lining. Some of these changes affected channel blockage. We tested the hypothesis that Asp172 – a residue implicated in channel gating by polyamines – formed an additional selectivity filter by using the triple mutant T141A/S165L/D172N. Large Rb+ and Cs+ currents were measured in this mutant. We propose that both Thr141 and Ser165 are likely to provide binding sites for monovalent blocking cations in wild‐type channels. These residues lie beyond the carbonyl oxygen tunnel thought to form the channel selectivity filter, which the blocking cations must therefore traverse.

The conformational changes associated with the interaction of sodium laurate with the recombinant heme domain for cytochrome P-450BM3 have been investigated by steady-state and picosecond-timeresolved fluorescence spectroscopy. The steady-state quenching experiments show that while all the five tryptophan residues are accessible to acrylamide in the free enzyme as well as the enzyme . substrate complex, the number of tryptophan residues accessible to ionic quenchers decreases on interaction of the substrate with the enzyme. This indicates that some of the tryptophan residues move towards the core of the protein on interaction with the substrate. The number of tryptophan residues accessible to the solvent as determined by the calculation of the solvent-accessible area for the free enzyme agrees with the values obtained by the quenching experiments.The time-resolved fluorescence studies carried out by means of the time-correlated single-photoncounting technique show that the fluorescence-decay curve is best fitted to a three-exponential model (0.2, 1.0 and 5.4 ns). Lifetime distributions, as recovered by the maximum-entropy method, agree with the discrete exponential model. The binding of the substrate does not lead to any significant change in the lifetime components of the enzyme, indicating that the tryptophan residues are possibly away from the substrate-binding domain. The decay-associated emission spectra and the magnitudes of amplitude of different lifetimes indicate that the shortest lifetime component ( 7 , ) originates from the three tryptophan residues that are completely or partially accessible to the solvent, and t, originates from the tryptophan residues that are buried in the core of the enzyme and not accessible to the solvent. X-ray crystallographic data and solvent-acessible-area calculations have been used to identify these residues.

The role of two cysteine residues--Cys122 and Cys154--in the structure of the strong inward rectifier K+ channel, Kir2.1, has been investigated using site-directed mutagenesis and electrophysiology. Such cysteine residues are conserved across the inward rectifier family and may be expected to form a crucial disulphide bond. Our experiments show that when the cysteines are absent, the protein is expressed, but the channels are not functional, suggesting that the disulphide bond is essential for correct channel assembly. However, reducing agents applied extracellularly have little effect on current amplitude in wild-type, so that, once the channel is assembled correctly in the membrane, the disulphide bonds are no longer essential for function. Molecular modelling suggests that a disulphide bond is formed--this may be either an intra- or an inter-subunit.

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