Cytochromes P450 (P450s) metabolize a wide range of endogenous compounds and xenobiotics, such as pollutants, environmental compounds, and drug molecules. The microsomal, membrane-associated, P450 isoforms CYP3A4, CYP2D6, CYP2C9, CYP2C19, CYP2E1, and CYP1A2 are responsible for the oxidative metabolism of more than 90% of marketed drugs. Cytochrome P450 3A4 (CYP3A4) metabolizes more drug molecules than all other isoforms combined. Here we report three crystal structures of CYP3A4: unliganded, bound to the inhibitor metyrapone, and bound to the substrate progesterone. The structures revealed a surprisingly small active site, with little conformational change associated with the binding of either compound. An unexpected peripheral binding site is identified, located above a phenylalanine cluster, which may be involved in the initial recognition of substrates or allosteric effectors.

https://science.sciencemag.org/content/sci/305/5684/683.full.pdf

Crystal Structures of Human Cytochrome P450 3A4 Bound to Metyrapone and Progesterone

Strategies for the prediction of in vivo drug clearance from in vitro drug metabolite kinetic data are well established for the rat. In this animal species, metabolism rate-substrate concentration relationships can commonly be described by the classic hyperbola consistent with the Michaelis-Menten model and simple scaling of the parameter intrinsic clearance (CL(int) - the ratio of V(max) to K(m)) is particularly valuable. The in vitro scaling of kinetic data from human tissue is more complex, particularly as many substrates for cytochrome P450 (CYP) 3A4, the dominant human CYP, show nonhyperbolic metabolism rate-substrate concentration curves. This review critically examines these types of data, which require the adoption of an enzyme model with multiple sites showing cooperative binding for the drug substrate, and considers the constraints this kinetic behavior places on the prediction of in vivo pharmacokinetic characteristics, such as metabolic stability and inhibitory drug interaction potential. The cases of autoactivation and autoinhibition are discussed; the former results in an initial lag in the rate-substrate concentration profile to generate a sigmoidal curve whereas the latter is characterized by a convex curve as V(max) is not maintained at high substrate concentrations. When positive cooperativity occurs, we suggest the use of CL(max), the maximal clearance resulting from autoactivation, as a substitute for CL(int). The impact of heteroactivation on this approach is also of importance. In the case of negative cooperativity, care in using the V(max)/K(m) approach to CL(int) determination must be taken. Examples of substrates displaying each type of kinetic behavior are discussed for various recombinant CYP enzymes, and possible artifactual sources of atypical rate-concentration curves are outlined. Finally, the consequences of ignoring atypical Michaelis-Menten kinetic relationships are examined, and the inconsistencies reported for both different substrates and sources of recombinant CYP3A noted.

In vitro-in vivo scaling of CYP kinetic data not consistent with the classical Michaelis-Menten model.

Comparisons of catalytic selectivity of cytochrome P450 subfamily enzymes from different species.

The cytochrome P450 enzymes are involved in the biotransformation of both xenobiotic and endobiotic hydrophobic compounds, implicated in the bioactivation of certain procarcinogens (e.g., benzo[ a ]pyrene), and responsible for many metabolism-based drug-drug interactions ([Wrighton and Stevens, 1992

Atypical Kinetic Profiles in Drug Metabolism Reactions

Cytochrome P450 3A4 is generally considered to be the most important human drug-metabolizing enzyme and is known to catalyze the oxidation of a number of substrates in a cooperative manner. An allosteric mechanism is usually invoked to explain the cooperativity. Based on a structure–activity study from another laboratory using various effector–substrate combinations and on our own studies using site-directed mutagenesis and computer modeling of P450 3A4, the most likely location of effector binding is in the active site along with the substrate. Our study was designed to test this hypothesis by replacing residues Leu-211 and Asp-214 with the larger Phe and Glu, respectively. These residues were predicted to constitute a portion of the effector binding site, and the substitutions were designed to mimic the action of the effector by reducing the size of the active site. The L211F/D214E double mutant displayed an increased rate of testosterone and progesterone 6β-hydroxylation at low substrate concentrations and a decreased level of heterotropic stimulation elicited by α-naphthoflavone. Kinetic analyses of the double mutant revealed the absence of homotropic cooperativity with either steroid substrate. At low substrate concentrations the steroid 6β-hydroxylase activity of the wild-type enzyme was stimulated by a second steroid, whereas L211F/D214E displayed simple substrate inhibition. To analyze L211F/D214E at a more mechanistic level, spectral binding studies were carried out. Testosterone binding by the wild-type enzyme displayed homotropic cooperativity, whereas substrate binding by L211F/D214E displayed hyperbolic behavior.

https://www.pnas.org/content/pnas/95/12/6636.full.pdf

Analysis of human cytochrome P450 3A4 cooperativity: construction and characterization of a site-directed mutant that displays hyperbolic steroid hydroxylation kinetics.

Analysis of Four Residues within Substrate Recognition Site 4 of Human Cytochrome P450 3A4: Role in Steroid Hydroxylase Activity and α-Naphthoflavone Stimulation

Juping Liu

Papers

Analysis of Four Residues within Substrate Recognition Site 4 of Human Cytochrome P450 3A4: Role in Steroid Hydroxylase Activity and α-Naphthoflavone Stimulation

Role of residue 480 in substrate specificity of cytochrome P450 2B5 and 2B11.