Positions of His‐64 and a bound water in human carbonic anhydrase II upon binding three structurally related inhibitors

The 3‐dimensional structure of human carbonic anhydrase II (HCAII; EC 4.2.1.1) complexed with 3 structurally related inhibitors, la, lb, and lc, has been determined by X‐ray crystallographic methods. The 3 inhibitors (la = C8H12N2O4S3) vary only in the length of the substituent on the 4‐amino group: la, proton; lb, methyl; and lc, ethyl. The binding constants (Ki's) for la, Ib, and lc to HCAII are 1.52, 1.88, and 0.37 nM, respectively. These structures were solved to learn if any structural cause could be found for the difference in binding. In the complex with inhibitors la and lb, electron density can be observed for His‐64 and a bound water molecule in the native positions. When inhibitor lc is bound, the side chain attached to the 4‐amino group is positioned so that His‐64 can only occupy the alternate position and the bound water is absent. While a variety of factors contribute to the observed binding constants, the major reason lc binds tighter to HCAII than does la or lb appears to be entropy: the increase in entropy when the bound water molecule is released contributes to the increase in binding and overcomes the small penalty for putting the His‐64 side chain in a higher energy state.