Mechanism of confinement enhancing enzyme intrinsic activity

doi:10.1016/S1872-2067(25)64827-3

Abstract

Abstract:

Enzymatic catalysis within surface- and volume-confined environments is common in biological cells or industrial applications. Despite their prevalence both in vivo and in vitro, a comprehensive mechanistic understanding of how these confinements tune the intrinsic activity of enzymes has remained elusive. Herein, we explore the role of confinement in shaping the activity of enzymes. Experiments show that the confinements induced by macromolecular crowding enhance lipase activity. To uncover the origin of the activity enhancement, thermodynamic activation parameters of lipase catalysis were calculated through extensive molecular dynamics (MD) and empirical valence bond (EVB) simulations. The EVB approach has proven to be an efficient method, enabling extensive sampling via MD and the evaluation of thermodynamic activation parameters for enzyme catalysis. Our findings reveal that confinement applied at the loop regions of lipase leads to higher intrinsic activities, and this effect depends on the degree of confinement. The lower free energy of activation originates from the gain of both enthalpy and entropy. Better preorganization of the active site and greater conformational space overlap between initial and transition states enhance lipase catalysis. We observe that the catalytic enhancement due to surface confinement is not exclusive to lipase but extends to PETase, highlighting its potential universality as a principle for enzyme design and engineering.

Key words: Enzyme catalysis, Surface confinement, Empirical valence bond calculation, Activation free energy, Enzyme design

Yufei Cao, Shuang Chen, Hui Liang, Junrong Yang, Wenyong Lou, Jun Ge. Mechanism of confinement enhancing enzyme intrinsic activity[J]. Chinese Journal of Catalysis, 2026, 81: 355-365.

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URL: https://www.cjcatal.com/EN/10.1016/S1872-2067(25)64827-3

https://www.cjcatal.com/EN/Y2026/V81/I2/355

Figures/Tables 6

Fig. 1. (a) Illustration of confined enzyme catalysis in vivo and in vitro. (b) Relative activity of BSLA under confinements by adding different polymer crowders at 25 and 40 °C. The enzyme catalysis in buffer solutions without crowders is labeled as "Control". The designations "PEG_1", "PEG_2", "PVP_1", "PVP_2", "Ficoll_1", and "Ficoll_2" represent the addition of PEG6k (polyethylene glycol, molecular weight: 6000) at 1 mg/L, PEG6k at 5 mg/L, PVP40k (polyvinylpyrrolidone, molecular weight: 40000) at 5 mg/L, PVP58k at 1 mg/L, Ficoll70 at 1 mg/L, and Ficoll70 at 2 mg/L, respectively.

Fig. 2. Impact of surface confinement on BSLA intrinsic activity. (a) Structures of the enzyme-substrate (ES) complex, transition state (TS), and enzyme-product (EP) complex. Substrate pNPB and catalytic triad (Ser77-His156-Asp133) were shown. (b) Calculated free energy profiles (298.15 K) from reactants to the tetrahedral intermediate in the acylation step for the reference reaction in water (blue) and BSLA (red). (c) Position distribution of the 11 lysine residues located on the surface of BSLA is shown. Additionally, the catalytic triad comprising Ser77-His156-Asp133 is indicated. (d) Activation free energy ΔG‡ calculations for BSLA under lysine restraint at 323.15 K are presented. The wild BSLA, without restraint, is represented as "wild type". The designation "rK44" denotes restraint (confinement) applied at residue Lys44, and so forth. "4_r" encompasses Lys44, Lys69, Lys112, and Lys122 under restraint. "6_r" encompasses Lys44, Lys69, Lys70, Lys95, Lys112, and Lys122 under restraint. Data is presented in kcal/mol-1 as the average values and standard error of the mean.

Fig. 3. The origin of reduced activation free energy in BSLA due to surface confinement. (a) Arrhenius plots depicting ΔG‡/T vs. 1/T for catalyzed reactions in wild type BSLA and the "6_r" BSLA variant. (b) A comparison of interatomic distances for four atom pairs within the transition states of wild BSLA and the "6_r" BSLA variant. (c) RMSF of both wild and confined BSLA at transition states. Dynamic structural ensembles of the ES and TS states for wild BSLA (d), and "6_r" BSLA variant (e), projected onto the eigenvectors corresponding to the two lowest-frequency principal components (PCA1 and PCA2).

Fig. 4. Rational confinement site selection for BSLA. (a) Differential ΔRMSF illustrating the disparity in conformational fluctuations between the ES and TS states of wild BSLA and the "6_r" variant. (b) Visualization of ΔRMSF of each residue. ΔRMSF values increase from blue to red. (c) Activation free energy ΔG‡ calculations for BSLA under restraint at 323.15 K are presented. The unrestrained wild-type BSLA is labeled as "wild type." The notation "rG13" signifies restraint (confinement) applied at residue Gly13, and so on. (d) Arrhenius plots depicting ΔG‡/T vs. 1/T for catalyzed reactions in wild type BSLA and the "rF16&S131" BSLA variant.

Fig. 5. Impact of confinements on the deacylation of BSLA. (a) The deacylation step of BSLA catalyzing pNPB hydrolysis. IT1 represents the acylated BSLA, IT2 is the tetrahedral intermediate, and PC is the final deacylated state. (b) Calculated free energy profiles (323.15 K) from the acylated BSLA (IT1) to the final deacylated state (PC) during the deacylation step for both wild-type BSLA and the confined variant "rF16&S131". The intermediate formed between the two transition states (TS1 and TS2) was designated as IT2. (c) Differential ΔRMSF illustrating the disparity in conformational fluctuations between the IT1 and TS2 states of wild-type BSLA.

Fig. 6. Confinement enhancing IsPETase activity. (a) Visualization of ΔRMSF of each residue. (b) Calculated activation free energy of wild and confined IsPETase at rationally selected sites at 310 K.

References

[1]	M. A. Huffman, A. Fryszkowska, O. Alvizo, M. Borra-Garske, K. R. Campos, K. A. Canada, P. N. Devine, D. Duan, J. H. Forstater, S. T. Grosser, H. M. Halsey, G. J. Hughes, J. Jo, L. A. Joyce, J. N. Kolev, J. Liang, K. M. Maloney, B. F. Mann, N. M. Marshall, M. McLaughlin, J. C. Moore, G. S. Murphy, C. C. Nawrat, J. Nazor, S. Novick, N. R. Patel, A. Rodriguez-Granillo, S. A. Robaire, E. C. Sherer, M. D. Truppo, A. M. Whittaker, D. Verma, L. Xiao, Y. Xu, H. Yang, Science, 2019, 366, 1255-1259.
[2]	H. Fu, J. Cao, T. Qiao, Y. Qi, S. J. Charnock, S. Garfinkle, T. K. Hyster, Nature, 2022, 610, 302-307.
[3]	J. Zhang, A. O. Maggiolo, E. Alfonzo, R. Mao, N. J. Porter, N. M. Abney, F. H. Arnold, Nat. Catal., 2023, 6, 152-160.
[4]	P. N. Devine, R. M. Howard, R. Kumar, M. P. Thompson, M. D. Truppo, N. J. Turner, Nat. Rev. Chem., 2018, 2, 409-421.
[5]	E. L. Bell, W. Finnigan, S. P. France, A. P. Green, M. A. Hayes, L. J. Hepworth, S. L. Lovelock, H. Niikura, S. Osuna, E. Romero, K. S. Ryan, N. J. Turner, S. L. Flitsch, Nat. Rev. Meth. Primers, 2021, 1, 46.
[6]	P. A. Romero, T. M. Tran, A. R. Abate, Proc. Natl. Acad. Sci. USA, 2015, 112, 7159-7164.
[7]	A. W. Senior, R. Evans, J. Jumper, J. Kirkpatrick, L. Sifre, T. Green, C. Qin, A. Žídek, A. W. R. Nelson, A. Bridgland, H. Penedones, S. Petersen, K. Simonyan, S. Crossan, P. Kohli, D. T. Jones, D. Silver, K. Kavukcuoglu, D. Hassabis, Nature, 2020, 577, 706-710.
[8]	J. Jumper, R. Evans, A. Pritzel, T. Green, M. Figurnov, O. Ronneberger, K. Tunyasuvunakool, R. Bates, A. Žídek, A. Potapenko, A. Bridgland, C. Meyer, S. A. A. Kohl, A. J. Ballard, A. Cowie, B. Romera-Paredes, S. Nikolov, R. Jain, J. Adler, T. Back, S. Petersen, D. Reiman, E. Clancy, M. Zielinski, M. Steinegger, M. Pacholska, T. Berghammer, S. Bodenstein, D. Silver, O. Vinyals, A. W. Senior, K. Kavukcuoglu, P. Kohli, D. Hassabis, Nature, 2021, 596, 583-589.
[9]	M. Baek, F. DiMaio, I. Anishchenko, J. Dauparas, S. Ovchinnikov, G. R. Lee, J. Wang, Q. Cong, L. N. Kinch, R. D. Schaeffer, C. Millan, H. Park, C. Adams, C. R. Glassman, A. DeGiovanni, J. H. Pereira, A. V. Rodrigues, A. A. van Dijk, A. C. Ebrecht, D. J. Opperman, T. Sagmeister, C. Buhlheller, T. Pavkov-Keller, M. K. Rathinaswamy, U. Dalwadi, C. K. Yip, J. E. Burke, K. C. Garcia, N. V. Grishin, P. D. Adams, R. J. Read, D. Baker, Science, 2021, 373, 871-876.
[10]	S. L. Lovelock, R. Crawshaw, S. Basler, C. Levy, D. Baker, D. Hilvert, A. P. Green, Nature, 2022, 606, 49-58.
[11]	V. Vaissier Welborn, T. Head-Gordon, Chem. Rev., 2019, 119, 6613-6630.
[12]	J. Wang, S. Lisanza, D. Juergens, D. Tischer, J. L. Watson, K. M. Castro, R. Ragotte, A. Saragovi, L. F. Milles, M. Baek, I. Anishchenko, W. Yang, D. R. Hicks, M. Expòsit, T. Schlichthaerle, J.-H. Chun, J. Dauparas, N. Bennett, B. I. M. Wicky, A. Muenks, F. DiMaio, B. Correia, S. Ovchinnikov, D. Baker, Science, 2022, 377, 387-394.
[13]	J. L. Watson, D. Juergens, N. R. Bennett, B. L. Trippe, J. Yim, H. E. Eisenach, W. Ahern, A. J. Borst, R. J. Ragotte, L. F. Milles, B. I. M. Wicky, N. Hanikel, S. J. Pellock, A. Courbet, W. Sheffler, J. Wang, P. Venkatesh, I. Sappington, S. V. Torres, A. Lauko, V. De Bortoli, E. Mathieu, S. Ovchinnikov, R. Barzilay, T. S. Jaakkola, F. DiMaio, M. Baek, D. Baker, Nature, 2023, 620, 1089-1100.
[14]	A. H. Yeh, C. Norn, Y. Kipnis, D. Tischer, S. J. Pellock, D. Evans, P. Ma, G. R. Lee, J. Z. Zhang, I. Anishchenko, B. Coventry, L. Cao, J. Dauparas, S. Halabiya, M. DeWitt, L. Carter, K. N. Houk, D. Baker, Nature, 2023, 614, 774-780.
[15]	A. Küchler, M. Yoshimoto, S. Luginbühl, F. Mavelli, P. Walde, Nat. Nanotechnol., 2016, 11, 409-420.
[16]	C. M. Agapakis, P. M. Boyle, P. A. Silver, Nat. Chem. Biol., 2012, 8, 527-535.
[17]	I. Wheeldon, S. D. Minteer, S. Banta, S. C. Barton, P. Atanassov, M. Sigman, Nat. Chem., 2016, 8, 299-309.
[18]	J. Fu, Y. R. Yang, A. Johnson-Buck, M. Liu, Y. Liu, N. G. Walter, N. W. Woodbury, H. Yan, Nat. Nanotechnol., 2014, 9, 531-536.
[19]	O. Idan, H. Hess, Nat. Nanotechnol., 2012, 7, 769-770.
[20]	O. Idan, H. Hess, ACS Nano, 2013, 7, 8658-8665.
[21]	G. A. Ellis, W. P. Klein, G. Lasarte-Aragonés, M. Thakur, S. A. Walper, I. L. Medintz, ACS Catal., 2019, 9, 10812-10869.
[22]	O. I. Wilner, Y. Weizmann, R. Gill, O. Lioubashevski, R. Freeman, I. Willner, Nat. Nanotechnol., 2009, 4, 249-254.
[23]	G. Ke, M. Liu, S. Jiang, X. Qi, Y. R. Yang, S. Wootten, F. Zhang, Z. Zhu, Y. Liu, C. J. Yang, H. Yan, Angew. Chem. Int. Ed., 2016, 55, 7483-7486.
[24]	C. J. Delebecque, A. B. Lindner, P. A. Silver, F. A. Aldaye, Science, 2011, 333, 470-474.
[25]	J. E. Dueber, G. C. Wu, G. R. Malmirchegini, T. S. Moon, C. J. Petzold, A. V. Ullal, K. L. J. Prather, J. D. Keasling, Nat. Biotechnol., 2009, 27, 753-759.
[26]	F. Lyu, Y. Zhang, R. N. Zare, J. Ge, Z. Liu, Nano Lett., 2014, 14, 5761-5765.
[27]	K. Liang, R. Ricco, C. M. Doherty, M. J. Styles, S. Bell, N. Kirby, S. Mudie, D. Haylock, A. J. Hill, C. J. Doonan, P. Falcaro, Nat. Commun., 2015, 6, 7240.
[28]	W.-H. Chen, M. Vázquez-González, A. Zoabi, R. Abu-Reziq, I. Willner, Nat. Catal., 2018, 1, 689-695.
[29]	W. Liang, P. Wied, F. Carraro, C. J. Sumby, B. Nidetzky, C.-K. Tsung, P. Falcaro, C. J. Doonan, Chem. Rev., 2021, 121, 1077-1129.
[30]	B. Panganiban, B. Qiao, T. Jiang, C. DelRe, M. M. Obadia, T. D. Nguyen, A. A. A. Smith, A. Hall, I. Sit, M. G. Crosby, P. B. Dennis, E. Drockenmuller, M. Olvera de la Cruz, T. Xu, Science, 2018, 359, 1239-1243.
[31]	C. DelRe, Y. Jiang, P. Kang, J. Kwon, A. Hall, I. Jayapurna, Z. Ruan, L. Ma, K. Zolkin, T. Li, C. D. Scown, R. O. Ritchie, T. P. Russell, T. Xu, Nature, 2021, 592, 558-563.
[32]	W. Liang, H. Xu, F. Carraro, N. K. Maddigan, Q. Li, S. G. Bell, D. M. Huang, A. Tarzia, M. B. Solomon, H. Amenitsch, L. Vaccari, C. J. Sumby, P. Falcaro, C. J. Doonan, J. Am. Chem. Soc., 2019, 141, 2348-2355.
[33]	R. Chapanian, D. H. Kwan, I. Constantinescu, F. A. Shaikh, N. A. A. Rossi, S. G. Withers, J. N. Kizhakkedathu, Nat. Commun., 2014, 5, 4683.
[34]	R. C. Rodrigues, C. Ortiz, Á. Berenguer-Murcia, R. Torres, R. Fernández-Lafuente, Chem. Soc. Rev., 2013, 42, 6290-6307.
[35]	X. Wu, H. Yue, Y. Zhang, X. Gao, X. Li, L. Wang, Y. Cao, M. Hou, H. An, L. Zhang, S. Li, J. Ma, H. Lin, Y. Fu, H. Gu, W. Lou, W. Wei, R. N. Zare, J. Ge, Nat. Commun., 2019, 10, 5165.
[36]	C. Hu, Y. Bai, M. Hou, Y. Wang, L. Wang, X. Cao, C.-W. Chan, H. Sun, W. Li, J. Ge, K. Ren, Sci. Adv., 2020, 6, eaax5785.
[37]	Y.-M. Li, J. Yuan, H. Ren, C.-Y. Ji, Y. Tao, Y. Wu, L.-Y. Chou, Y.-B. Zhang, L. Cheng, J. Am. Chem. Soc., 2021, 143, 15378-15390.
[38]	F. Secundo, Chem. Soc. Rev., 2013, 42, 6250.
[39]	J. Ge, J. Lei, R. N. Zare, Nat. Nanotechnol., 2012, 7, 428-432.
[40]	Y. Chen, F. Jiménez-Ángeles, B. Qiao, M. D. Krzyaniak, F. Sha, S. Kato, X. Gong, C. T. Buru, Z. Chen, X. Zhang, N. C. Gianneschi, M. R. Wasielewski, M. Olvera de la Cruz, O. K. Farha, J. Am. Chem. Soc., 2020, 142, 18576-18582.
[41]	K. Kawasaki, H. Kondo, M. Suzuki, S. Ohgiya, S. Tsuda, Acta Crystallogr. Sect. D Biol. Crystallogr., 2002, 58, 1168-1174.
[42]	S. Joo, I. J. Cho, H. Seo, H. F. Son, H.-Y. Sagong, T. J. Shin, S. Y. Choi, S. Y. Lee, K.-J. Kim, Nat. Commun., 2018, 9, 382.
[43]	M. H. M. Olsson, C. R. Søndergaard, M. Rostkowski, J. H. Jensen, J. Chem. Theory Comput., 2011, 7, 525-537.
[44]	P. Mark, L. Nilsson, J. Phys. Chem. A, 2001, 105, 9954-9960.
[45]	G. A. Kaminski, R. A. Friesner, J. Tirado-Rives, W. L. Jorgensen, J. Phys. Chem. B, 2001, 105, 6474-6487.
[46]	M. Purg, M. Elias, S. C. L. Kamerlin, J. Am. Chem. Soc., 2017, 139, 17533-17546.
[47]	M. Biler, R. M. Crean, A. K. Schweiger, R. Kourist, S. C. L. Kamerlin, J. Am. Chem. Soc., 2020, 142, 20216-20231.
[48]	C. I. Bayly, P. Cieplak, W. Cornell, P. A. Kollman, J. Phys. Chem., 1993, 97, 10269-10280.
[49]	G. King, A. Warshel, J. Chem. Phys., 1989, 91, 3647-3661.
[50]	P. Bauer, A. Barrozo, M. Purg, B. A. Amrein, M. Esguerra, P. B. Wilson, D. T. Major, J. Åqvist, S. C. L. Kamerlin, SoftwareX, 2018, 7, 388-395.
[51]	F. S. Lee, A. Warshel, J. Chem. Phys., 1992, 97, 3100-3107.
[52]	H. J. C. Berendsen, J. P. M. Postma, W. F. van Gunsteren, A. DiNola, J. R. Haak, J. Chem. Phys., 1984, 81, 3684-3690.
[53]	J.-P. Ryckaert, G. Ciccotti, H. J. C. Berendsen, J. Comput. Phys., 1977, 23, 327-341.
[54]	R. T. McGibbon, K. A. Beauchamp, M. P. Harrigan, C. Klein, J. M. Swails, C. X. Hernández, C. R. Schwantes, L.-P. Wang, T. J. Lane, V. S. Pande, Biophys. J., 2015, 109, 1528-1532.
[55]	A. Shkurti, R. Goni, P. Andrio, E. Breitmoser, I. Bethune, M. Orozco, C. A. Laughton, SoftwareX, 2016, 5, 44-50.
[56]	W. Humphrey, A. Dalke, K. Schulten, J. Mol. Graph., 1996, 14, 33-38.
[57]	J. S. Weltz, D. F. Kienle, D. K. Schwartz, J. L. Kaar, J. Am. Chem. Soc., 2020, 142, 3463-3471.
[58]	H. Sanchez-Moran, J. L. Kaar, D. K. Schwartz, Nat. Commun., 2024, 15, 2299.
[59]	J. Åqvist, A. Warshel, Chem. Rev., 1993, 93, 2523-2544.
[60]	M. Purg, S. C. L. Kamerlin, Methods Enzymol, 2018, 607, 3-51.
[61]	P. Acharya, E. Rajakumara, R. Sankaranarayanan, N. M. Rao, J. Mol. Biol., 2004, 341, 1271-1281.
[62]	S. Ahmad, M. Z. Kamal, R. Sankaranarayanan, N. M. Rao, J. Mol. Biol., 2008, 381, 324-340.
[63]	E. A. Hoyt, P. M. S. D. Cal, B. L. Oliveira, G. J. L. Bernardes, Nat. Rev. Chem., 2019, 3, 147-171.
[64]	K. Maruyama, T. Ishiyama, Y. Seki, K. Sakai, T. Togo, K. Oisaki, M. Kanai, J. Am. Chem. Soc., 2021, 143, 19844-19855.
[65]	J. Kim, B. X. Li, R. Y. C. Huang, J. X. Qiao, W. R. Ewing, D. W. C. MacMillan, J. Am. Chem. Soc., 2020, 142, 21260-21266.
[66]	G. V. Isaksen, J. Åqvist, B. O. Brandsdal, PLoS Comput. Biol., 2014, 10, e1003813.
[67]	S. A. Hindson, H. A. Bunzel, B. Frank, D. A. Svistunenko, C. Williams, M. W. van der Kamp, A. J. Mulholland, C. R. Pudney, J. L. Ross Anderson, ACS Catal., 2021, 11, 11532-11541.
[68]	F. van der Ent, B. A. Lund, L. Svalberg, M. Purg, G. Chukwu, M. Widersten, G. V. Isaksen, B. O. Brandsdal, J. Åqvist, Biochemistry, 2022, 61, 933-942.
[69]	S. Yoshida, K. Hiraga, T. Takehana, I. Taniguchi, H. Yamaji, Y. Maeda, K. Toyohara, K. Miyamoto, Y. Kimura, K. Oda, Science, 2016, 351, 1196-1199.
[70]	C. Jerves, R. P. P. Neves, M. J. Ramos, S. da Silva, P. A. Fernandes, ACS Catal., 2021, 11, 11626-11638.
[71]	R. García-Meseguer, E. Ortí, I. Tuñón, J. J. Ruiz-Pernía, J. Aragó, J. Am. Chem. Soc., 2023, 145, 19243-19255.
[72]	S. Feng, Y. Yue, M. Zheng, Y. Li, Q. Zhang, W. Wang, ACS Sustainable Chem. Eng., 2021, 9, 9823-9832.