STRUCTURAL-ANALYSIS OF THE ZINC HYDROXIDE-THR-199-GLU-106 HYDROGEN-BOND NETWORK IN HUMAN CARBONIC ANHYDRASE-II

The significance of the zinc hydroxide-Thr-199-Glu-106 hydrogen-bond n etwork in the active site of human carbonic anhydrase II has been exam ined by X-ray crystallographic analyses of site-specific mutants. Muta nts with Ala-199 and Ala-106 or Gln-106 have low catalytic activities, while a mutant with Asp-106 has almost full CO2 hydration activity. T he structures of these four mutants, as well as that of the bicarbonat e complex of the mutant with Ala-199, have been determined at 1.7 to 2 .2 angstrom resolution. Removal of the gamma atoms of residue 199 lead s to a distorted tetrahedral geometry at the zinc ion, and a catalytic ally important zinc-bound water molecule has moved towards Glu-106. In the bicarbonate complex of the mutant with Ala-199 one oxygen atom fr om bicarbonate binds to zinc without displacing this water molecule. T etrahedral coordination geometries are retained in the mutants at posi tion 106. The mutants with Ala-106 and Gln-106 have a zinc-bound sulfa te ion, whereas this sulfate site is only partially occupied in the mu tant with Asp-166. The hydrogen-bond network seems to be ''reversed'' in the mutants with Ala-106 and Gln-106. The network is preserved as i n native enzyme in the mutant with Asp-106 but the side chain of Asp-1 06 is more extended than that of Glu-106 in the native enzyme. These r esults illustrate the importance of Glu-106 and Thr-199 for controllin g the precise coordination geometry of the zinc ion and its ligand pre ferences which results in an optimal orientation of a zinc-bound hydro xide ion for an attack on the CO2 substrate. (C) 1993 Wiley-Liss, Inc.