Human CYP2E1 accounts for almost 2% of total CYP enzymes in the liver cells, and plays a crucial role in the metabolism of
small molecular weight compounds. This enzyme is associated with the nearly 6% metabolisms of the currently clinical drugs. However,
it is found that CYP2E1 has a non-hyperbolic kinetic profile that can not be explained by the common Michaelis-Menten mechanism.
Further studies show that the non-hyperbolic kinetic behaviors are associated with multiple substrate binding, which is also known as the
cooperative binding properties. However, the detailed mechanism for the cooperative binding is not clear by now. In this paper, we summarized
the experimental and theoretical studies on the cooperative binding mechanism. Based on the structural analysis, a second substrate
binding site is confirmed in human CYP2E1, which is located neither in the region near Leu103, Leu210 and Phe478, nor far from
the active site. Additionally, two important residues Thr303 and Phe478 are also identified to be the key factors in the cooperative binding
on the short-range and long-range effects, respectively. The former plays a crucial role in the positioning of substrates and in proton
delivery to the active site; the latter is located between the substrate access channel and the active site, and exhibits directly effects on
substrate access or on substrate positioning in the active site. All these points can provide useful information for the cooperative binding
in human CYP2E1, revealing the detailed mechanism for the non-hyperbolic kinetic behaviors.