Substrate access channel geometry of soluble and membrane-bound cytochromes P450 as studied by interactions with type II substrate analogues

Krainev, Arkadii G.; Weiner, Lev; Kondrashin, Sergey K.; Kanaeva, Irina P.; Bachmanova, G.I.

doi:10.1016/0003-9861(91)90159-g

Cited by 12 publications

(6 citation statements)

References 8 publications

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“…Therefore, alterations of the structure of the distal site caused by the Glu318 -* Ala mutation may be subsequent effects on the protein molecule. Results obtained here are in good agreement with suggestions derived from X-ray crystal structure (Poulos et al, 1985 and alignments of amino acid sequences of P450s (Nelson & Strobel, 1988Gotoh & Fujii-Kuriyama, 1989) in that the whole protein structure of membrane-bound P450 may be similar to that of bacterial 450 (Krainev et al, 1991).…”

Section: Resultssupporting

confidence: 89%

“…It is known that mammalian P450s isolated from microsomal membranes associate themselves in the aqueous solution like other integral membrane proteins (Sato & Omura, 1978;Golly et al, 1988). P450 exists in the monomeric state at a concentration of Emulgen 913 more than 0.026% (w:v) (Bachmanova et al, 1989;Hildebrandt et al, 1989a;Krainev et al, 1991).…”

Section: Resultsmentioning

confidence: 99%

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Absorption spectral study of cytochrome P450d-phenyl isocyanide complexes: effects of mutations at the putative distal site on the conformational stability

Krainev

Shimizu²,

Ishigooka³

et al. 1991

Biochemistry

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Interactions of phenyl isocyanide (PheNC) with purified engineered cytochrome P450d wild type and putative distal mutants, Glu318Asp and Glu318Ala, were studied with optical absorption spectra. The wild type and the mutant Glu318Asp were purified as the high-spin state, while the mutant Glu318Ala was purified as the oxygen-bound low-spin form. Thus, it is suggested that Glu318 is important to make the appropriate heme environment of P450d. Spectral dissociation constants (0.19-0.39 mM) of the ligand for the ferric mutants were lower than that (0.74 mM) of the wild type. These dissociation constants were changed by adding a substrate, 7-ethoxycoumarin. The reduced wild type-PheNC complex showed a Soret peak at 451 nm, while the reduced mutant-PheNC complexes showed two peaks at 451 and 423 nm. The 451-nm peak of the complexes decreased with the concomitant increase of a new peak at 433 nm at room temperature. Thus, it was suggested that P450d can take two conformationally different forms from the characteristic spectral features. The Soret spectral conversions which followed the first-order kinetics were analyzed by changing the temperature. The activation energy (69 kcal/mol) for the conversion for the wild type was higher than those (37-50 kcal/mol) for the mutants. The activation energy for the wild type further increased (by 55%) by adding the substrate, while those for the mutants were essentially unchanged by adding the substrate. We discuss the important role of Glu318 at the putative distal site of P450d in the packing or the conformational stability of the putative distal site of the P450d molecule.

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Section: Resultssupporting

confidence: 89%

Section: Resultsmentioning

confidence: 99%

Absorption spectral study of cytochrome P450d-phenyl isocyanide complexes: effects of mutations at the putative distal site on the conformational stability

Krainev

Shimizu²,

Ishigooka³

et al. 1991

Biochemistry

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“…A surprising result was obtained subsequent to mutation Lys250Leu at one of the putative nonactive site residues (Gotoh, 1992;Krainev et al, 1991bKrainev et al, , 1992. We observe that this mutation also enhanced the chiral discrimination of this enzyme (Tables Hand III).…”

Section: Resultsmentioning

confidence: 61%

“…Lys250 is conjectured to be located at one of the putative substrate-recognition sites of eukaryotic P450s and is thought to be remote from the active site (Gotoh, 1992;Krainev et al, 1991bKrainev et al, , 1992. The Lys250Leu mutant bound with the three chiral axial ligands formed nitrogen-bound low-spin complexes (Table I).…”

Section: Resultsmentioning

confidence: 99%

“…Thus, Lys250 seems to be equally important in the chiral discrimination of external axial ligands as has been observed for Glu318 in this enzyme. It was conjectured from optical absorption spectral studies by using bifunctional axial ligands that Lys250 and neighboring ionic amino acids are located at the entrance of the substrate-(or ligand-) access channel of microsomal P450s (Krainev et al, 1991b). In fact, mutations of Lys250 and neighboring ionic amino acids to neutral ones in P450 1A2 enhanced catalytic activities by 3-6-fold toward methoxyresorufin and ethoxyresorufln (Krainev et al, 1992).…”

Section: Discussionmentioning

confidence: 99%

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Chiral recognition at cytochrome P450 1A2 active site: Effects of mutations at the putative distal site on the bindings of asymmetrical axial ligands

Krainev¹,

Shimizu²,

Ishigooka³

et al. 1993

Biochemistry

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Effects of mutations at the putative distal site of cytochrome P450 1A2 on chiral discrimination for binding (R)-(+)- and (S)-(-)-1-(1-naphthyl)ethylamine (ligand I), (R)-(-)- and (S)-(+)-1-cyclohexylethylamine (ligand II), and (R)-(+)- and (S)-(-)-1-(4-pyridyl)ethanol (ligand III) were studied by optical absorption spectra. The wild-type P450 1A2 exhibited different dissociation constants (Kd) for the R- and S-enantiomers of these ligands. The R/S ratios of the Kd values for ligands I and II were 5.2 and 2.9, respectively, and the S/R ratio for ligand III was 6.0. Mutations at the putative distal site, such as Glu318Asp and Glu318Ala, remarkably enhanced the discrimination: the R/S ratio of the Kd values for ligand I increased from 5.2 to 20-60, while the R/S ratio for ligand II decreased from 2.9 to 0.8-0.9. These remarkable changes in the R/S ratios were not observed with Glu318Asp mutation for ligand III binding, whereas affinities for both enantiomers of ligand III were markedly decreased by the Glu318Ala mutation. Mutation Thr319Ala increased the R/S ratio of the Kd values for ligand I slightly but markedly decreased the R/S ratio of ligand II (from 2.9 to 0.8) and the S/R ratio of ligand III (from 6.0 to 1.0). Similar enhancements of the chiral discriminations were observed with the mutation Lys250Leu at another putative substrate-recognition site. Differences between the R- and S-enantiomers of the standard enthalpy and entropy of ligand III binding were changed most remarkably by the Thr319Ser mutation.(ABSTRACT TRUNCATED AT 250 WORDS)

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Cytochrome P450 Reactions in the Human Brain

Haining¹

2007

Handbook of Neurochemistry and Molecular Neurobiology

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Substrate access channel geometry of soluble and membrane-bound cytochromes P450 as studied by interactions with type II substrate analogues

Cited by 12 publications

References 8 publications

Absorption spectral study of cytochrome P450d-phenyl isocyanide complexes: effects of mutations at the putative distal site on the conformational stability

Absorption spectral study of cytochrome P450d-phenyl isocyanide complexes: effects of mutations at the putative distal site on the conformational stability

Chiral recognition at cytochrome P450 1A2 active site: Effects of mutations at the putative distal site on the bindings of asymmetrical axial ligands

Cytochrome P450 Reactions in the Human Brain

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