Mechanistic and Kinetic Insights into the Acylation Reaction of Hepatitis C Virus NS3/NS4A Serine Protease with NS4B/5A Substrate

Martínez-González, José Ángel; Salazar-Sanchez, Nuria; Larriva-Hormigos, María; Martínez, Rodrigo; González, Miguel

doi:10.3390/biom15111619

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Mechanistic and Kinetic Insights into the Acylation Reaction of Hepatitis C Virus NS3/NS4A Serine Protease with NS4B/5A Substrate

by

José Ángel Martínez-González

^1,2,*

,

Nuria Salazar-Sanchez

³

,

María Larriva-Hormigos

¹,

Rodrigo Martínez

²

and

Miguel González

⁴

¹

Departamento de Química y Bioquímica, Facultad de Farmacia, Universidad San Pablo-CEU, CEU Universities, Urbanización Montepríncipe, Boadilla del Monte, 28668 Madrid, Spain

²

Departamento de Química, Universidad de La Rioja, C/Madre de Dios, 51, 26006 Logroño, Spain

³

Departamento de Ciencias Farmacéuticas y de la Salud, Facultad de Farmacia, Universidad San Pablo-CEU, CEU Universities, Urbanización Montepríncipe, Boadilla del Monte, 28668 Madrid, Spain

⁴

Departament de Ciència de Materials i Química Física and IQTC, Universitat de Barcelona, C/Martí i Franquès, 1, 08028 Barcelona, Spain

^*

Author to whom correspondence should be addressed.

Biomolecules 2025, 15(11), 1619; https://doi.org/10.3390/biom15111619

Submission received: 14 July 2025 / Revised: 8 September 2025 / Accepted: 14 November 2025 / Published: 18 November 2025

(This article belongs to the Section Chemical Biology)

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Abstract

Reaction mechanisms and rate constants of the acylation reaction of the hepatitis C virus (HCV) NS3/NS4A serine protease with the NS4B/5A natural substrate were studied using SCC-DFTB/MM (self-consistent charge density functional tight binding/molecular mechanics) and EA-VTST/MT (ensemble-averaged variational transition state theory/multidimensional tunneling) methods, considering the isotope effect (H/D). This reaction is crucial in the HCV life cycle. The reaction follows an essentially concerted mechanism. Although two elementary steps are involved, no intermediate step has been found between them. Thus, the proposed general two-step serine protease acylation mechanism, which includes a tetrahedral intermediate, does not occur here. This finding aligns with our studies on another natural substrate (NS5A/5B), indicating a greater variety in mechanism than previously expected. Tunneling and recrossing play an intermediate role; the activation free energy barriers are in good agreement with the experimental value, and the kinetic isotope effect (k(H)/k(D)) is somewhat larger than one (1.3). The rate constant value is not reproduced due to the exponential dependence of the rate constant on the activation free energy.

Keywords: hepatitis C virus; enzyme catalysis; concerted mechanism in serine protease reaction; NS3/NS4A serine protease; QM/MM reaction mechanism; EA-VTST/MT; rate constants; substrateNS4B/5A

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MDPI and ACS Style

Martínez-González, J.Á.; Salazar-Sanchez, N.; Larriva-Hormigos, M.; Martínez, R.; González, M. Mechanistic and Kinetic Insights into the Acylation Reaction of Hepatitis C Virus NS3/NS4A Serine Protease with NS4B/5A Substrate. Biomolecules 2025, 15, 1619. https://doi.org/10.3390/biom15111619

AMA Style

Martínez-González JÁ, Salazar-Sanchez N, Larriva-Hormigos M, Martínez R, González M. Mechanistic and Kinetic Insights into the Acylation Reaction of Hepatitis C Virus NS3/NS4A Serine Protease with NS4B/5A Substrate. Biomolecules. 2025; 15(11):1619. https://doi.org/10.3390/biom15111619

Chicago/Turabian Style

Martínez-González, José Ángel, Nuria Salazar-Sanchez, María Larriva-Hormigos, Rodrigo Martínez, and Miguel González. 2025. "Mechanistic and Kinetic Insights into the Acylation Reaction of Hepatitis C Virus NS3/NS4A Serine Protease with NS4B/5A Substrate" Biomolecules 15, no. 11: 1619. https://doi.org/10.3390/biom15111619

APA Style

Martínez-González, J. Á., Salazar-Sanchez, N., Larriva-Hormigos, M., Martínez, R., & González, M. (2025). Mechanistic and Kinetic Insights into the Acylation Reaction of Hepatitis C Virus NS3/NS4A Serine Protease with NS4B/5A Substrate. Biomolecules, 15(11), 1619. https://doi.org/10.3390/biom15111619

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Mechanistic and Kinetic Insights into the Acylation Reaction of Hepatitis C Virus NS3/NS4A Serine Protease with NS4B/5A Substrate

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