Revealing insights into the catalytic mechanism of the Soybean Lipoxygenase-1 using quantum mechanics/molecular mechanics (QM/MM) calculations. This study focuses on the effect of substrate and inhibitor molecules on the catalytic mechanism of soybean lipoxygenase-1 (SLO-1). The catalytic activity of SLO-1 can be inhibited by 3,5-di-tert-butyl catechol (DTBC), which has a structure similar to linoleic acid (LA). In order to understand the catalytic mechanism of SLO-1, the initial conformation of DTBC, the LA/DTBC model, was constructed by energy minimization. In addition, other possible DTBC molecules with altered molecular conformations were also constructed. For the purpose of validating the validity of DTBC for the LA/DTBC model, a series of simulations were performed using a reduced model that was constructed by excluding the LA molecule from the LA/DTBC model. First, six snapshots of the catalytic trajectory were taken from the corresponding umbrella sampling simulation of the LA/DTBC model, and a reaction path was extracted using the maximum likelihood algorithm. In the subsequent molecular dynamics simulations and QM/MM calculations, the average structure of the snapshots obtained from the simulation of the LA/DTBC model was used as the initial conformation for modeling the DTBC/SLO-1 system. According to the QM/MM calculations, DTBC can enter the SLO-1 active site through the channel and bind to the side chains of key residues in the active site via hydrogen bonds and hydrophobic interactions. DTBC is well positioned and in close proximity to the catalytic residues, where it can enhance the catalytic reaction. More importantly, DTBC has a larger distance between its two hydroxyl groups than that between the two hydroxyl groups of LA, which may result in decreased electron density in the aromatic ring. This leads to a reduced ability to donate electrons to the substrate. Therefore, DTBC and LA may have distinct mechanisms of electron transfer. This finding may provide some explanation for the lower activity of SLO-1 with the addition of DTBC. It is hypothesized that the inhibition mechanism of SLO-1 against DTBC is due to the competition between DTBC and LA, which may lead to the reduced ability of SLO-1 to catalyze unsaturated fatty acids. These results will help to reveal the molecular mechanism of the inhibition effect and catalyzing reactions of SLO-1. In addition, this research provides some new theoretical insight into the design of inhibitors that may have inhibitory effects on SLO-1.