Mapping of cytochrome P450 2B4 substrate binding sites by photolabile probe 3-azidiamantane: Identification of putative substrate access regions

2007 ◽  
Vol 468 (1) ◽  
pp. 82-91 ◽  
Author(s):  
Petr Hodek ◽  
Martin Karabec ◽  
Miroslav Šulc ◽  
Bruno Sopko ◽  
Stanislav Smrček ◽  
...  
Keyword(s):  
Cytochrome P450 ◽  
Binding Sites ◽  
Substrate Binding ◽  
Cytochrome P450 2B4 ◽  
Substrate Binding Sites

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

Xenobiotica ◽  
2010 ◽  
Vol 41 (4) ◽  
pp. 281-289 ◽  
Author(s):  
Harshica Fernando ◽  
Jessica A. O. Rumfeldt ◽  
Nadezhda Y. Davydova ◽  
James R. Halpert ◽  
Dmitri R. Davydov
Keyword(s):  
Cytochrome P450 ◽  
Binding Sites ◽  
Substrate Binding ◽  
Cytochrome P450 3A4 ◽  
Multiple Substrate ◽  
Substrate Binding Sites

scholarly journals Sigmoidal kinetic model for two co-operative substrate-binding sites in a cytochrome P450 3A4 active site: an example of the metabolism of diazepam and its derivatives

1999 ◽  
Vol 340 (3) ◽  
pp. 845-853 ◽  
Author(s):  
Magang SHOU ◽  
Qin MEI ◽  
JR. Michael W. ETTORE ◽  
Renke DAI ◽  
Thomas A. BAILLIE ◽  
...  
Keyword(s):  
Cytochrome P450 ◽  
Kinetic Model ◽  
Binding Affinity ◽  
Active Site ◽  
Binding Sites ◽  
Dissociation Constants ◽  
Substrate Binding ◽  
Cytochrome P450 3A4 ◽  
Multiple Drugs ◽  
Substrate Binding Sites

Cytochrome P450 3A4 (CYP3A4) plays a prominent role in the metabolism of a vast array of drugs and xenobiotics and exhibits broad substrate specificities. Most cytochrome P450-mediated reactions follow simple Michaelis-Menten kinetics. These parameters are widely accepted to predict pharmacokinetic and pharmacodynamic consequences in vivo caused by exposure to one or multiple drugs. However, CYP3A4 in many cases exhibits allosteric (sigmoidal) characteristics that make the Michaelis constants difficult to estimate. In the present study, diazepam, temazepam and nordiazepam were employed as substrates of CYP3A4 to propose a kinetic model. The model hypothesized that CYP3A4 contains two substrate-binding sites in a single active site that are both distinct and co-operative, and the resulting velocity equation had a good fit with the sigmoidal kinetic observations. Therefore, four pairs of the kinetic estimates (KS1, kα, KS2, kβ, KS3, k∆, KS4 and kγ) were resolved to interpret the features of binding affinity and catalytic ability of CYP3A4. Dissociation constants KS1 and KS2 for two single-substrate-bound enzyme molecules (SE and ES) were 3-50-fold greater than KS3 and KS4 for a two-substrate-bound enzyme (SES), while respective rate constants k∆ and kγ were 3-218-fold greater than kα and kβ, implying that access and binding of the first molecule to either site in an active pocket of CYP3A4 can enhance the binding affinity and reaction rate of the vacant site for the second substrate. Thus our results provide some new insights into the co-operative binding of two substrates in the inner portions of an allosteric CYP3A4 active site.

Substrate-binding sites in acetylcholinesterase

1991 ◽  
Vol 12 ◽  
pp. 422-426 ◽  
Author(s):  
Ferdinand Hucko ◽  
Jaak Järv ◽  
Christoph Weise
Keyword(s):  
Binding Sites ◽  
Substrate Binding ◽  
Substrate Binding Sites

scholarly journals Human organic anion transporter MRP4 (ABCC4) is an efflux pump for the purine end metabolite urate with multiple allosteric substrate binding sites

2005 ◽  
Vol 288 (2) ◽  
pp. F327-F333 ◽  
Author(s):  
Rémon A. M. H. Van Aubel ◽  
Pascal H. E. Smeets ◽  
Jeroen J. M. W. van den Heuvel ◽  
Frans G. M. Russel
Keyword(s):  
Binding Sites ◽  
Efflux Pump ◽  
Substrate Binding ◽  
Organic Anion ◽  
Apical Cell ◽  
Cell Uptake ◽  
Hek293 Cells ◽  
Hill Coefficient ◽  
Anion Transporter ◽  
Substrate Binding Sites

The end product of human purine metabolism is urate, which is produced primarily in the liver and excreted by the kidney through a well-defined basolateral blood-to-cell uptake step. However, the apical cell-to-urine efflux mechanism is as yet unidentified. Here, we show that the renal apical organic anion efflux transporter human multidrug resistance protein 4 (MRP4), but not apical MRP2, mediates ATP-dependent urate transport via a positive cooperative mechanism ( Km of 1.5 ± 0.3 mM, Vmax of 47 ± 7 pmol·mg−1·min−1, and Hill coefficient of 1.7 ± 0.2). In HEK293 cells overexpressing MRP4, intracellular urate levels were lower than in control cells. Urate inhibited methotrexate transport (IC50 of 235 ± 8 μM) by MRP4, did not affect cAMP transport, whereas cGMP transport was stimulated. Urate shifted cGMP transport by MRP4 from positive cooperativity ( Km and Vmax value of 180 ± 20 μM and 58 ± 4 pmol·mg−1·min−1, respectively, Hill coefficient of 1.4 ± 0.1) to single binding site kinetics ( Km and Vmax value of 2.2 ± 0.9 mM and 280 ± 50 pmol·mg−1·min−1, respectively). Finally, MRP4 could transport urate simultaneously with cAMP or cGMP. We conclude that human MRP4 is a unidirectional efflux pump for urate with multiple allosteric substrate binding sites. We propose MRP4 as a candidate transporter for urinary urate excretion and suggest that MRP4 may also mediate hepatic export of urate into the circulation, because of its basolateral expression in the liver.

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