Spacer-Based Selectivity in the Binding of “Two-Prong” Ligands to Recombinant Human Carbonic Anhydrase I

• Published in: Biochemistry (Easton) Vol. 44; no. 9; pp. 3211 - 3224
• Main Authors: Banerjee, Abir L, Eiler, Daniel, Roy, Bidhan C, Jia, Xiao, Haldar, Manas K, Mallik, Sanku, Srivastava, D. K
• Format: Journal Article
• Language: English
• Published: United States American Chemical Society 08.03.2005
• Subjects: Acetazolamide - chemistry; Acetazolamide - metabolism; Benzenesulfonamides; Binding, Competitive; Calorimetry; Carbonic Anhydrase I - antagonists & inhibitors; Carbonic Anhydrase I - metabolism; Carbonic Anhydrase Inhibitors - chemistry; Carbonic Anhydrase Inhibitors - metabolism; Dansyl Compounds - metabolism; Diethyl Pyrocarbonate - chemistry; Humans; Imino Acids - chemical synthesis; Imino Acids - metabolism; Kinetics; Ligands; Protein Binding; Recombinant Proteins - antagonists & inhibitors; Recombinant Proteins - metabolism; Spectrometry, Fluorescence; Sulfonamides - chemical synthesis; Sulfonamides - metabolism; Titrimetry
• ISSN: 0006-2960; 1520-4995
• DOI: 10.1021/bi047737b

Benzenesulfonamide and iminodiacetate (IDA)-conjugated Cu2+ independently interact at the active site and a peripheral site of carbonic anhydrases, respectively [Banerjee, A. L., Swanson, M., Roy, B. C., Jia, X., Haldar, M. K., Mallik, S., and Srivastava, D. K. (2004) J. Am. Chem. Soc. 126, 10875−10883]. By attaching IDA-bound Cu2+ to benzenesulfonamide via different chain length spacers, we synthesized two “two-prong” ligands, L1 and L2, in which the distances between Cu2+ and NH2 group of sulfonamide were 29 and 22 Å, respectively. We compared the binding affinities of L1 and L2, vis-à-vis their parent compound, benzenesulfonamide, for recombinant human carbonic anhydrase I (hCA-I) by performing the fluorescence titration and steady-state kinetic experiments. The experimental data revealed that whereas the binding affinity of L1 for hCA-I was similar to that of benzenesulfonamide, the binding affinity of L2 was ∼2 orders of magnitude higher, making L2 one of the most potent ligands or inhibitors of hCA-I. Since the enhanced binding or inhibitory potency of L2 is diminished (to the level of benzenesulfonamide) either in the presence of EDTA or upon treatment of the enzyme with diethyl pyrocarbonate, it is proposed that Cu2+ of L2 interacts with one of the surface-exposed histidine residues of the enzyme. A cumulative account of the experimental data leads to the suggestion that the differential binding of L1 versus L2 to hCA-I is encoded in the chain length of the spacer moiety.