Binding of 1-(4-chloro-benzenesulfonyl)-4-hydroxy-pyrrolidine-2-carboxylic acid to human carbonic anhydrase II affects its structure and function: a multi-spectroscopic and molecular docking study.

The interaction between human carbonic anhydrase II (hCA II) and 1-(4-chloro-benzenesulfonyl)-4-hydroxy-pyrrolidine-2-carboxylic acid (CBHPCA) was inspected by applying different spectroscopic methods and molecular docking, as well. Kinetic studies revealed that CBHPCA inhibits hCA II esterase activity through a linear competitive mode. Fluorescence analyses showed that CBHPCA quenches the enzyme's intrinsic fluorescence through a static quenching mechanism. Thermodynamic analyses indicated that hydrogen bonding and van der Waals interactions stabilize the CBHPCA-enzyme complex. Protein surface hydrophobicity (PSH) index of the enzyme reduced upon CBHPCA interaction, as shown by a fluorometric study using 1-anilinonaphthalene-8-sulfonic acid. Oxidation studies with N-boromosuccinimide (NBS) revealed a decrease in the number of attainable tryptophans of the enzyme in the presence of CBHPCA. Far-UV circular dichroism (CD) spectroscopy indicated a rise in the α-helicity of hCA II upon CBHPCA binding, while near-UV CD spectra showed decreased flexibility of the enzyme's tertiary structure. Acrylamide quenching experiments demonstrated reduced accessibility of tryptophans of the enzyme to the quencher, consistent with NBS experiment results. Guanidine hydrochloride denaturation experiments showed increased thermodynamic stability of the CBHPCA-enzyme complex compared to the enzyme alone. Fourier transform infrared experiments confirmed some increment in the α-helicity of the enzyme induced by CBHPCA. Molecular docking study revealed that hydrogen bonding and van der Waals interactions caused anchoring of CBHPCA to the enzyme's active site. It seems that structural compactness induced by CBHPCA binding to the enzyme may be accountable for its increased thermodynamic stability.