Structural changes of cellobiohydrolase I (1,4-beta-D-glucan-cellobiohydrolase I, CBHI) and PNPC (p-nitrophenyl-beta-D-cellobioside) during the binding process.
Conformational changes to 1,4-beta-D-glucan cellobiohydrolase I (CBHI) in response to its binding with p-nitrophenyl beta-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.
Wu
Bin
B
State Key Laboratory of Microbial Technology, Shandong University, Jinan 250100, China.
Wang
LuShan
L
Gao
PeiJi
P
D002236:Carbohydrate Conformation; D043366:Cellulose 1,4-beta-Cellobiosidase / Q000737:chemistry* / Q000378:metabolism; D005960:Glucosides / Q000737:chemistry* / Q000378:metabolism; D011485:Protein Binding; D011487:Protein Conformation; D013050:Spectrometry, Fluorescence; D013056:Spectrophotometry, Ultraviolet
D005960:Glucosides; C034170:4-nitrophenyl beta-cellobioside; D043366:Cellulose 1,4-beta-Cellobiosidase