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Over the years the proteasome has emerged as a crucial target for the treatment of many diseases such as neurodegenerative diseases, cancer, autoimmune diseases, developmental disorders, cystic fibrosis, diabetes, cardiac diseases, atherosclerosis, and aging. In this work, the mechanism of proteasome covalent inhibition with bisbenzyl-protected homobelactosin C (hBelC) was explored using quantum mechanics\/molecular mechanics (QM\/MM) methods. Molecular dynamic simulations were used to describe key interactions established between the hBelC and its unique binding mode in the primed site of the \u03b25 subunit. The free energy surfaces were computed to characterize the kinetics and thermodynamics of the inhibition process. This study revealed that although the final inhibition product for hBelC is formed according to the same molecular mechanism as one described for hSalA, the free energy profile of the reaction pathway differs significantly from the one previously reported for \u03b3-lactam-\u03b2-lactone containing inhibitors in terms of the height of the activation barrier as well as the stabilization of the final product. Moreover, it was proved that high stabilization of the covalent adduct formed between \u03b25-subunit and hBelC, together with the presence of aminocarbonyl side chain in the structure of the inhibitor which prevents the hydrolysis of the ester bond from taking place, determines its irreversible character.<\/jats:p>","DOI":"10.3390\/ph15050531","type":"journal-article","created":{"date-parts":[[2022,4,26]],"date-time":"2022-04-26T02:14:39Z","timestamp":1650939279000},"page":"531","update-policy":"https:\/\/doi.org\/10.3390\/mdpi_crossmark_policy","source":"Crossref","is-referenced-by-count":5,"title":["Covalent Inhibition of the Human 20S Proteasome with Homobelactosin C Inquired by QM\/MM Studies"],"prefix":"10.3390","volume":"15","author":[{"given":"Natalia","family":"Serrano-Aparicio","sequence":"first","affiliation":[{"name":"BioComp Group, Department of Physical and Analytical Chemistry, Universitat Jaume I, 12071 Castell\u00f3n de la Plana, Spain"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Silvia","family":"Ferrer","sequence":"additional","affiliation":[{"name":"BioComp Group, Department of Physical and Analytical Chemistry, Universitat Jaume I, 12071 Castell\u00f3n de la Plana, Spain"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0002-7528-1551","authenticated-orcid":false,"given":"Katarzyna","family":"\u015awiderek","sequence":"additional","affiliation":[{"name":"BioComp Group, Department of Physical and Analytical Chemistry, Universitat Jaume I, 12071 Castell\u00f3n de la Plana, Spain"}],"role":[{"vocabulary":"crossref","role":"author"}]}],"member":"1968","published-online":{"date-parts":[[2022,4,25]]},"reference":[{"key":"ref_1","unstructured":"Hyams, J.S. 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