POSITIONS OF HIS-64 AND A BOUND WATER IN HUMAN CARBONIC-ANHYDRASE-II UPON BINDING 3 STRUCTURALLY RELATED INHIBITORS

The 3-dimensional structure of human carbonic anhydrase II (HCAII; EC 4.2.1.1) complexed with 3 structurally related inhibitors, 1a, 1b, and 1c, has been determined by X-ray crystallographic methods. The 3 inhi bitors (1a = C8H12N2O4S3) vary only in the length of the substituent o n the 4-amino group: 1a, proton; 1b, methyl; and 1c, ethyl. The bindin g constants (K-i's) for 1a, 1b, and 1c to HCAII are 1.52, 1.88, and 0. 37 nM, respectively. These structures were solved to learn if any stru ctural cause could be found for the difference in binding. In the comp lex with inhibitors 1a and 1b, electron density can be observed for Hi s-64 and a bound water molecule in the native positions. When inhibito r 1c is bound, the side chain attached to the 4-amino group is positio ned so that His-64 can only occupy the alternate position and the boun d water is absent. While a variety of factors contribute to the observ ed binding constants, the major reason 1c binds tighter to HCAII than does 1a or 1b appears to be entropy: the increase in entropy when the bound water molecule is released contributes to the increase in bindin g and overcomes the small penalty for putting the His-64 side chain in a higher energy state.