PROTON-TRANSFER IN THE CATALYTIC MECHANISM OF CARBONIC-ANHYDRASE .2. EFFECTS OF PLACING HISTIDINE-RESIDUES AT VARIOUS POSITIONS IN THE ACTIVE-SITE OF HUMAN ISOENZYME-II

The maximal rate of CO2 hydration catalyzed by human carbonic anhydras e II (carbonate hydro-lyase, EC 4.2.1.1) is limited by proton transfer steps involving the acid-base function of His-64. To test whether or not the precise location of this proton transfer group is critical, hi stidine residues were placed in various positions in the active site o f the enzyme. Thus, four double mutants were made, all with His-64 rep laced by Ala-64, and with a histidine residue replacing Asn-62, Ala-65 , Asn-67 or Thr-200. The results show that the mutants with His-62, Hi s-67 and His-200, but not the mutant with His-65, yield significantly higher k(cat) values for CO2 hydration than the single mutant with Ala -64, indicating that His-62, His-67 and His-200 can contribute to prot on transfer between the metal center and the reaction medium. However, the average proton transfer efficiency of these histidines is only ab out 5% of that of His-64 in the unmodified enzyme.