Correlations between spin equilibrium shift, reduction rate, and N-demethylation activity in liver microsomal cytochrome P-450 and a series of benzphetamine analogues as substrates

J, Blanck; Rein, H.; Sommer, Manfred; Ristau, O.; Smettan, G; Ruckpaul, K

doi:10.1016/0006-2952(83)90109-0

Cited by 46 publications

(16 citation statements)

References 20 publications

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“…Similar thermal effects on membrane-bound P450 have been also documented with other substrates [106]. In all instances examined, there was reasonable relationship between the substrate-induced degree of high-spin transition of the heme iron, hydroxylase activity and/or rate/proportion of NADPH-supported P450 reduction in the burst phase [104,107,108]. On the other hand, there did not appear to exist a strict dependence of the catalytic efficiency (V max /K m ) of biotransformation of a series of benzphetamines on the percentage high-spin enzyme [104].…”

Section: Studies With Impure P450 Systemssupporting

confidence: 61%

“…In all instances examined, there was reasonable relationship between the substrate-induced degree of high-spin transition of the heme iron, hydroxylase activity and/or rate/proportion of NADPH-supported P450 reduction in the burst phase [104,107,108]. On the other hand, there did not appear to exist a strict dependence of the catalytic efficiency (V max /K m ) of biotransformation of a series of benzphetamines on the percentage high-spin enzyme [104]. Judging from the stoichiometry between rate of electron transfer to ferric P450 and product formation from ethylmorphine, demethylation of the N-heterocycle was concluded to be a tightly coupled reaction despite moderate affinity (K d = 59 μM) of the compound for the microsomal hemoprotein [109].…”

Section: Studies With Impure P450 Systemsmentioning

confidence: 79%

“…Addition of hexobarbital and other type I substrates to the buffered suspensions disclosed the magnitude of A max , measured at the 385/420 nm wavelength pair in the difference spectra as a reflection of spinstate changes in the microsomal P450 system, to increase with decreasing K d ; spin equilibria turned out to be sensitive to pH modulation [103]. In contrast, examination of a test set of benzphetamine analogues failed to demonstrate correlation between K d values and spin shift [104], but provided clear evidence of susceptibility of the latter quantity to temperature variation [105]. Similar thermal effects on membrane-bound P450 have been also documented with other substrates [106].…”

Section: Studies With Impure P450 Systemsmentioning

confidence: 90%

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Control by Substrate of the Cytochrome P450-Dependent Redox Machinery: Mechanistic Insights

Hlavica¹

2007

CDM

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Based on initial studies with bacterial CYP101A1, a popular concept emerged predicting that substrate-induced low-to-high spin conversion of P450s is universally associated with shifts of the midpoint potential to a more positive value to maximize rates of electron transfer and metabolic turnover. However, evaluation of the plethora of observations with pro- and eukaryotic hemoproteins suggests a caveat as to generalization of this principle. Thus, some P450s are inherently high-spin, so that there is no need for a supportive substrate-triggered impulse to electron flow. With other enzymes, high-spin content is not consonant with reductive activity, and spin transition as such is not essential to sustaining substrate oxidation. Also, with certain proteins the low-spin conformer is reduced as swift as the high-spin entity. Moreover, there is not regularly a linear relationship between high-spin level and anodic shift of the reduction potential. Similarly, in given cases turnover may proceed despite insignificant or even lacking substrate-provoked alterations in the redox behaviour. Thus, folding of the disparate and sometimes conflicting data into a harmonized overall picture is a lingering problem. Apart from direct perturbation of the electrochemical properties, substrate docking may entail changes in enzyme conformation such as to favour productive complexation with redox partners or modulate electron transfer conduits within preformed donor/acceptor adducts, resulting in elevated ease of flow of reducing equivalents. Substrate-steered ordering of the oligomeric aggregation state of P450s is likely to impose steric constraints on heterodimers, causing one component to more readily align with electron carriers. Careful uncovering of electrochemical mechanisms in these systems will be fruitful to tailoring of novel bioenergetic machines and redox chains via redox-inspired protein engineering or molecular Lego, capable of generating products of interest or degrading toxic pollutants. Finally, availability of P450 nanobiochips for high-throughput screening of substrate libraries might expedite drug development.

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Section: Studies With Impure P450 Systemssupporting

confidence: 61%

Section: Studies With Impure P450 Systemsmentioning

confidence: 79%

Section: Studies With Impure P450 Systemsmentioning

confidence: 90%

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Control by Substrate of the Cytochrome P450-Dependent Redox Machinery: Mechanistic Insights

Hlavica¹

2007

CDM

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“…Although the interrelationship between the substrate-induced spin shift and the efficiency of P450-catalysis is not straightforward, a conversion from the hexa-coordinated low-spin to the penta-coordinated high-spin state has an important impact on the catalytic efficiency and coupling of the P450 (see (Hlavica, 2007) for review). This link is demonstrated clearly, for example, in studies with CYP2B4 and a series of benzphetamine analogs (Blanck et al ., 1983; Schwarze et al ., 1985; Blanck et al , 1991). Those results reveal a strict correlation between the rate of substrate N -demethylation and its coupling to NADPH oxidation with the amplitude of the substrate-induced spin shift.…”

Section: Discussionmentioning

confidence: 80%

Multiple substrate-binding sites are retained in cytochrome P450 3A4 mutants with decreased cooperativity

Fernando¹,

Rumfeldt²,

Davydova³

et al. 2010

Xenobiotica

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Abstract1. The basis of decreased cooperativity in substrate binding in the cytochrome P450 3A4 mutants F213W, F304W and L211F/D214E was studied with fluorescence resonance energy transfer (FRET) and absorbance spectroscopy.2. Whereas in the wild type enzyme the absorbance changes reflecting the interactions with 1-pyrenebutanol exhibit a Hill coefficient (n H ) around 1.7 (S 50 = 11.7 μM), the mutants showed no cooperativity (n H ≤ 1.1) with unchanged S 50 values.3. Contrary to the premise that the mutants lack one of the two binding sites, the mutants exhibited at least two substrate binding events. The high affinity interaction is characterized by a dissociation constant (K D ) ≤ 1.0 μM, whereas the K D of the second binding has the same magnitude as the S 50 .4. Theoretical analysis of a two-step binding model suggests that n H values may vary from 1.1 to 2.2 depending on the amplitude of the spin shift caused by the first binding event.5. Alteration of cooperativity in the mutants is caused by a partial displacement of the "spinshifting" step. Whereas in the wild type the spin shift occurs in the ternary complex only, the mutants exhibit some spin shift upon binding of the first substrate molecule.

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“…So far several drugs are known to produce substrate binding spectra [25]. The affinity of the drugs to the P-450 varies [26] and at least in some cases correlates with their rate of metabolism [27].…”

Section: Discussionmentioning

confidence: 99%

Purification and characterization of a liver microsomal cytochrome P‐450 isoenzyme with a high affinity and metabolic capacity for coumarin from pyrazole‐treated D2 mice

Juvonen

Shkumatov

Lang

1988

European Journal of Biochemistry

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Microsomal coumarin 7-hydroxylase activity is regulated differently from several other monooxygenase enzymes, at least in mice [Wood, A. W. and Conney, A. H. (1974) Science (Wash. DC) 612-6141. Recently we found that in D2 mice this activity is strongly and selectively induced by pyrazole [Juvonen, R. O., Kaipainen, P. K. and Lang, M. A. (1985) Eur. J . Biochem. 152,. This paper describes the purification of the pyrazoleinducible cytochrome P-450 isoenzyme. Because of the lability of the protein, a special procedure for the purification was developed. The procedure is based on a combination of hydrophobic and ion-exchange chromatography and the presence of 100 FM coumarin in the preparations throughout the whole purification.Coumarin effectively protected the P-450 from degradation and also converted the pyrazole-inducible P-450 to its high-spin state. This enabled us to choose only those fractions for further purification where the P-450(s) was in its high-spin state (rather than measuring the content of the total P-450). As a result the purified protein had an apparent molecular mass of 49.7 kDa, a specific content of 19.9 nmol/mg protein and a very high affinity and metabolic capacity for coumarin.It has been suggested that the Coh locus regulates the 7-hydroxylation of coumarin (Coh for coumarin hydroxylase). This is based on the fact that the liver microsomal coumarin 7-hydroxylase activity varies among inbred strains of mice and is differently regulated from other liver microsomal P-450-dependent reactions [l]. A high microsomal coumarin 7-hydroxylase activity has also been found in rabbits, guinea-pigs, hamsters, pigs and humans [2-41. As a structural gene product of this locus, a so-far unknown cytochrome P-450 isoenzyme, capable of catalyzing the 7-hydroxylation of coumarin, was suggested to exist [5]. In 1985 Kaipainen et al. [6] from our laboratory gave the first direct evidence for the existence of this protein by purifying a cytochrome P-450 isoenzyme from liver microsomes of phenobarbital-pretreated D2 mice with a high affinity and specificity for coumarin 7-hydroxylation.Recently we found [7] that microsomal coumarin 7-hydroxylase is strongly increased in mice by pyrazole, a compound better known as an inhibitor of alcohol dehydrogenase. This induction is interesting in at least three different respects. First, the effect of pyrazole is much stronger than that of phenobarbital, the only inducer so far known to induce coumarin 7-hydroxylase. Second, the induction was strong only in D2 mice and weak or absent in B6 or AKR mice. Third, the effect on microsomal P-450-catalyzed reactions was selective, unlike that of phenobarbital, since only coumarin 7-hydroxylation (and to a lesser extent 7-ethoxycoumarin 0-deethylase) was increased by the compound while several other P-450-Correspondence to M. A. Lang, Farmakologian Laitos ja Toksikologian Laitos, Kuopion Yliopisto, Postilokero 6, SF-7021 1 Kuopio, Finland dependent reactions, in addition to the total microsomal P-450 content, were either not affe...

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Correlations between spin equilibrium shift, reduction rate, and N-demethylation activity in liver microsomal cytochrome P-450 and a series of benzphetamine analogues as substrates

Cited by 46 publications

References 20 publications

Control by Substrate of the Cytochrome P450-Dependent Redox Machinery: Mechanistic Insights

Control by Substrate of the Cytochrome P450-Dependent Redox Machinery: Mechanistic Insights

Multiple substrate-binding sites are retained in cytochrome P450 3A4 mutants with decreased cooperativity

Purification and characterization of a liver microsomal cytochrome P‐450 isoenzyme with a high affinity and metabolic capacity for coumarin from pyrazole‐treated D2 mice

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