Allosteric P450 mechanisms: multiple binding sites, multiple conformers or both?

Abstract

According to the initial hypothesis on the mechanisms of cooperativity in drug-metabolizing cytochromes P450, a loose fit of a single substrate molecule in the P450 active site results in a requirement for the binding of multiple ligand molecules for efficient catalysis. Although simultaneous occupancy of the active site by multiple ligands is now well established, there is increasing evidence that the mechanistic basis of cooperativity also involves an important ligandinduced conformational transition. Moreover, recent studies demonstrate that the conformational heterogeneity of the enzyme is stabilized by ligand-dependent interactions of several P450 molecules. Application of the concept of an oligomeric allosteric enzyme to microsomal cytochromes P450 in combination with a general paradigm of multiple ligand occupancy of the active site provides an excellent explanation for complex manifestations of the atypical kinetic behavior of the enzyme.

Chain length-dependent cooperativity in fatty acid binding and oxidation by cytochrome P450BM3 (CYP102A1)

“…The heme distal pocket and the narrow substrate access channel in P450 BM3 are unlikely to accommodate more than two palmitate molecules, indicating an allosteric rather than space-filling mechanism (Davydov and Halpert, 2008) for at …”

“…Numerous examples of sigmoidal kinetics in P450 activity have been described since the first report of such behavior in androstenedione oxidation by rabbit CYP2B enzymes (Ingelman-Sundberg and Johansson, 1980). Atypical kinetics is particularly common for mammalian CYP3A4 and CYP1A2 (Davydov and Halpert, 2008;Denisov et al, 2009), but is also found in bacterial enzymes such as CYP107A1 and CYP102A2/A3. Two substrate binding sites have been established on the distal side of the heme in CYP3A4, but the binding of up to six substrate molecules, together with surface binding sites, open conformations and equilibration between oligomers, have been proposed to model the complex behavior (Davydov and Halpert, 2008).…”

“…Atypical kinetics is particularly common for mammalian CYP3A4 and CYP1A2 (Davydov and Halpert, 2008;Denisov et al, 2009), but is also found in bacterial enzymes such as CYP107A1 and CYP102A2/A3. Two substrate binding sites have been established on the distal side of the heme in CYP3A4, but the binding of up to six substrate molecules, together with surface binding sites, open conformations and equilibration between oligomers, have been proposed to model the complex behavior (Davydov and Halpert, 2008). The effect of changes at sites remote from the active site on the product selectivity of P450 BM3 was noted in the oxidation of non-natural substrates by the R47L/Y51F mutant (Carmichael and Wong, 2001), and allosteric effects as well as space-filling mechanisms have been proposed for atypical kinetics in drug oxidation by the R47L/F87A/L188Q mutant (van Vugt-Lussenburg et al, 2006).…”

Analysis of Heterotropic Cooperativity in Cytochrome P450 3A4 Using α-Naphthoflavone and Testosterone

“…The most abundant of these in the adult human liver is cytochrome P450 3A4 (CYP3A4) 2 (1), which metabolizes approximately half of the most commonly prescribed drugs (2). Its ability to interact simultaneously with multiple substrate molecules leads to atypical kinetic phenomena, termed homotropic or heterotropic cooperativity (3)(4)(5)(6), the former describing interactions of molecules of the same substrate and the latter referring to interactions of different substrates. Evidence of CYP3A4 simultaneously interacting with multiple substrate molecules comes from the crystal structures of the enzyme, which show a large, plastic active site (7), substrate bound at a peripheral binding site (8), changes in kinetic behavior in the presence of so-called effector molecules (9 -12), as well as a global analysis of multiple observable enzyme properties (13)(14)(15)(16).…”

Organization of Cytochrome P450 Enzymes Involved in Sex Steroid Synthesis

“…The phenomenon of cooperativity in drug-metabolizing P450s involves simultaneous binding of multiple ligands that influence overall catalytic activity (11,12), as well as direct P450-P450 interactions (4,13). P450s catalyzing sex steroid synthesis, with much limited substrate specificities are unlikely to be able to bind or exchange multiple ligands in the way drug-metabolizing P450s can.…”