HECT-containing E3 proteins with a small number in human are directly involved in catalysis: Ub is transferred from E2 to the catalytic cysteine of the HECT domain, and then to a target lysine residue. RING family members represent most of the E3s, of which there are over 600 encoded in the human genome. They possess a conserved arrangement of cysteine and histidine residues that coordinate two zinc atoms. Unlike HECT E3, RING E3 ligases function as substrate recognition factors and mediate direct transfer of Ub from E2 to a lysine on the substrate or Ub itself, creating a polyubiquitin chain. Small ubiquitin-like modifier modifies proteins posttranslationally. SUMO-targeted ubiquitin ligases are a conserved family of proteins that target SUMO-modified proteins for ubiquitylation and typified by RING finger protein 4 in mammals. Four SUMO interaction motifs in the N-terminal region of RNF4 allow it to engage polySUMO-modified substrates. A RING domain in the Cterminal region is responsible for dimerization and catalysis of Ub transfer. RNF4 plays a key role in DNA damage response, accurate chromosome segregation during MK-0683 mitosis, and arsenic therapy for acute promyelocytic leukaemia. It also regulates the localization and function of the HTLV-1 oncoprotein Tax that promotes cell survival during reduced oxygen conditions, as well as other pathophysiological conditions. Ubiquitination can be outlined in the following steps: E2,Ub changes conformation when bounds to a RING E3 ligase. In this way, the thioester bond linking E2 and Ub is highly activated. An incoming substrate lysine is deprotonated and acts as a nucleophile for the E2,Ub thioester bond, followed by the substrate binding. How RING E3s promote Ub transfer remains unclear. Three major mechanisms have been proposed for lysine deprotonation: i) a local microenvironment which reduces the substrate lysine pK, ii) the optimal position of the incoming lysine e-amino group and reactive thioester bond, and iii) an acidic residue attracts the proton from the e-amino group. These suspects are only based on structures and activity analysis from kinetic and mutational biochemical experiments. No theoretical studies were found to describe intermediates and associated energy barriers in the reaction pathway. Therefore, our endeavors turn to understanding transition state points and reaction energy barriers, which may apply to regulators of ubiquitin ligase enzymes. Taken together, the elaborate elucidation of ubiquitin transfer catalysis is not only of great fundamental interest, but also of high medical relevance. Thus, we investigated the catalytic mechanism of Ub transfer by combining molecular modeling, MD simulations, and QM/MM calculations. Two different substrate binding models of RNF4 RING-UbcH5A-Ub-SUMO2 in aqueous solution were obtained from an MD simulation. The most proper model was chosen to probe the catalytic mechanism of proton transfer and nucleophilic attack. Our simulation results highlight the role of residues D117 and N77, which are consistent with experiment studies. These findings provide an atomic description of Ub transfer including mechanisms for substrate lysine deprotonation and nucleophilic.