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Structural changes of cellobiohydrolase I (1,4-β-D-glucan-cellobiohydrolase I, CBHI) and PNPC (p-nitrophenyl-β-D-cellobioside) during the binding process

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Abstract

Conformational changes to 1,4-β-D-glucan cellobiohydrolase I (CBHI) in response to its binding with p-nitrophenyl β-D-cellobioside (PNPC) were analyzed by second-derivative fluorescence spectrometry at the saturation binding point. Irreversible changes to the configuration of PNPC during the course of the binding process were characterized by UV spectral analysis. Isothermal titration calorimetry (ITC) was used to determine the stoichiometry of binding (i.e. the number of molar binding sites) of PNPC to CBHI. Two points on the surface of the CBHI molecule interact with PNPC, and irreversible changes to the configuration of PNPC occur during its conversion to p-nitrophenyl (PNP). The ITC studies demonstrated that the binding of PNPC to CBHI is an irreversible process, in which heat is released, but where there is no reversible equilibrium between PNPC-CBHI and CBHI and PNPC. On the other hand, PNP and cellobiose need to be released from the PNPC-CBHI complex to facilitate the repeated binding of new PNPC molecules to the renewable CBHI molecules. Therefore, we speculate that the energy, which powers the configurational change of PNPC as it is converted to PNP, is generated from cyclic changes in the conformation of CBHI during the binding/de-sorption process. These new insights may provide a basis for a better understanding of the binding mechanism in enzyme-substrate interactions.

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Authors and Affiliations

  1. State Key Laboratory of Microbial Technology, Shandong University, Jinan, 250100, China

    Bin Wu, LuShan Wang & PeiJi Gao

  2. Institute of Poultry Science, Shandong Academy of Agricultural Sciences, Jinan, 250023, China

    Bin Wu

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Supported by the National Natural Science Foundation of China (Grant Nos. 30370013 and 30500007)

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Wu, B., Wang, L. & Gao, P. Structural changes of cellobiohydrolase I (1,4-β-D-glucan-cellobiohydrolase I, CBHI) and PNPC (p-nitrophenyl-β-D-cellobioside) during the binding process. SCI CHINA SER C 51, 459–469 (2008). https://doi.org/10.1007/s11427-008-0064-2

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