Systematic Investigation of CYP3A4 Using Side-by-Side Comparisons of Apo, Active Site, and Allosteric-Bound States.

Abstract

Cytochrome P450 (CYP) 3A4 (CYP3A4) is a complex enzyme that metabolizes diverse substrates. It contains a large binding site accommodating diverse ligands, binding to active or allosteric sites. CYP3A4 does not always follow Michaelis-Menten kinetics. While K_m reflects substrate affinity, it does not necessarily determine the enzyme's activity, though it is often considered indicative of substrate binding characteristics. The mechanism may be highly sophisticated and driven by multiple factors. This suggests that the ligand binding affinity alone may not explain the differential behavior of the enzyme conformational stability. Here, we analyzed sequence conserveness of 57 CYPs, followed by a detailed molecular dynamics simulation study (9 μs) on CYP3A4. We studied three CYP3A4 enzyme states (apo-state, active-site, and allosteric-site ligand-bound states) collected from the same experimental setup to reduce the systematic error. We found that the enzyme conformational stability followed a consistent trend of allosteric > active > apo states, which was inconsistent with the enzyme-ligand (active/allosteric) binding affinity and the ligand conformational stability. However, the heme group showed a significant protein affinity and stability pattern directly related to the enzyme stability, suggesting that the active/allosteric binding may work by influencing the heme-CYP3A4 binding affinity, and the allosteric ligand appeared to form the most stable enzyme state of the three studied states.