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Benzylsuccinate synthase (BSS) catalyzes the enantiospecific formation of (R)-benzylsuccinate from toluene and fumarate, initiating anaerobic toluene degradation. In this paper, we present the first microkinetic analysis of the full reaction, predicting kinetic isotope effects in the range of 2.587\u20132.604, close to the experimentally observed value (KIEexp= 2.13 \u00b1 0.1). Our experiments confirmed that KIE values are lower in direct assays with substrate-saturated enzyme, relative to the values obtained for competitive kinetic isotope effects D(V\/K) via substrate fractionation (3.69 \u00b1 0.16). We postulate that this apparent KIE suppression originates from preferential binding of unlabeled versus labeled substrates to the enzyme, as well as from the rates of product release. We also show that tunneling effects have only minor influence on the observed KIE and D(V\/K) values, although they have been estimated to potentially accelerate the overall reaction rate by approximately 17%. On the other hand, inclusion in the kinetic equation of the barrier recrossing effect, jointly with tunneling correction can result in a lowering of predicted KIE values to the range of 2.38\u20132.43. Furthermore, we analyze a slowly occurring, experimentally observed H\/D exchange process in the product during incubation in D2O, confirming partial reversibility of the reaction. We estimate the rate of this H\/D exchange and propose a potential mechanism. Our study contributes to elucidating the processes catalyzed by BSS and its role in the bioremediation of hydrocarbon pollutants.<\/jats:p>","DOI":"10.1021\/acs.jpcb.6c01533","type":"journal-article","created":{"date-parts":[[2026,9,2]],"date-time":"2026-09-02T01:03:17Z","timestamp":1788310997000},"page":"9045-9057","source":"Crossref","is-referenced-by-count":0,"title":["The Origin of the\nKinetic Isotope Effect and Mechanism\nof H\/D Exchange in Radical C\u2013C Bond Formation Catalyzed by\nBenzylsuccinate Synthase: Experiments and Microkinetic Modeling"],"prefix":"10.1021","volume":"130","author":[{"ORCID":"https:\/\/orcid.org\/0000-0002-7650-9263","authenticated-orcid":true,"given":"Maciej","family":"Szaleniec","sequence":"first","affiliation":[{"name":"Polish Academy of Sciences , , ,","place":["Krako\u0301w, Poland, 30-239"]}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Gabriela","family":"Oleksy","sequence":"additional","affiliation":[{"name":"Polish Academy of Sciences , , ,","place":["Krako\u0301w, Poland, 30-239"]},{"name":"Philipps University Marburg , , ,","place":["Marburg, Germany, 35043"]}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Ivana","family":"Aleksic","sequence":"additional","affiliation":[{"name":"Polish Academy of Sciences , , ,","place":["Krako\u0301w, Poland, 30-239"]}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Kai","family":"Kra\u0308mer","sequence":"additional","affiliation":[{"name":"Philipps University Marburg , , ,","place":["Marburg, Germany, 35043"]}],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0002-3450-3576","authenticated-orcid":true,"given":"Tomasz","family":"Borowski","sequence":"additional","affiliation":[{"name":"Polish Academy of Sciences , , ,","place":["Krako\u0301w, Poland, 30-239"]}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Johann","family":"Heider","sequence":"additional","affiliation":[{"name":"Philipps University Marburg , , ,","place":["Marburg, Germany, 35043"]},{"name":"SYNMIKRO Research Center , ,","place":["Marburg, Germany, 35043"]}],"role":[{"vocabulary":"crossref","role":"author"}]}],"member":"316","published-online":{"date-parts":[[2026,8,28]]},"reference":[{"issue":"31","key":"2026091008082061200_cit1","doi-asserted-by":"publisher","first-page":"7688","DOI":"10.1021\/jp014627v","article-title":"Catalytic\nmechanism of benzylsuccinate synthase, a theoretical study","volume":"106","author":"Himo","year":"2002","journal-title":"J. Phys. Chem. B"},{"issue":"46","key":"2026091008082061200_cit2","doi-asserted-by":"publisher","first-page":"13932","DOI":"10.1021\/bi061117o","article-title":"Deuterium isotope effects in the unusual addition of\ntoluene to fumarate catalyzed by benzylsuccinate synthase","volume":"45","author":"Li","year":"2006","journal-title":"Biochemistry"},{"issue":"6","key":"2026091008082061200_cit3","doi-asserted-by":"publisher","first-page":"3361","DOI":"10.1021\/acscatal.0c04954","article-title":"Determinants for Substrate Recognition in the Glycyl\nRadical Enzyme Benzylsuccinate Synthase Revealed by Targeted Mutagenesis","volume":"11","author":"Salii","year":"2021","journal-title":"ACS Catal."},{"key":"2026091008082061200_cit4","doi-asserted-by":"publisher","first-page":"9","DOI":"10.1016\/j.jinorgbio.2014.05.006","article-title":"Mechanistic basis for the enantioselectivity\nof the anaerobic hydroxylation\nof alkylaromatic compounds by ethylbenzene dehydrogenase","volume":"139","author":"Szaleniec","year":"2014","journal-title":"J. Inorg. Biochem."},{"issue":"24","key":"2026091008082061200_cit5","doi-asserted-by":"publisher","first-page":"5823","DOI":"10.1021\/acs.jpcb.4c01237","article-title":"Modeling the Initiation Phase of\nthe Catalytic Cycle in the Glycyl-Radical Enzyme Benzylsuccinate Synthase","volume":"128","author":"Szaleniec","year":"2024","journal-title":"J. Phys. Chem. 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