Carbon- 13 Nuclear Magnetic Resonance as a Probe of Side Chain Orientation and Mobility in Carboxymethylated Human Carbonic Anhydrase BT

boxymethyl His-200 human carbonic anhydrase B have been obtained as a function of pH and in the presence and absence of the active site Zn(I1) or Cd(I1) ion. Chemical shifts of the I-l3C show that the carboxyl is sensitive to two ionization processes, with apparent pK,s of 7.2 and 9.9, respectively. These are assigned to the deprotonation of the N " of His-200 and the breaking of the coordination bond between the car-boxyl oxygen and the Zn(I1) ion, respectively. Assignment of the lower pK, to that of the N " is supported by the observation of this same ionization in the chemical shift of the 2-I3C res-onance showing the signal from the methylene carbon to un-dergo the same upfield shift as is observed on the ionization of the N " in NT-carboxymethylated histidine. The high pH ion-ization process is not reflected in the resonance of the meth-ylene carbon. No changes in the chemical shifts vs. pH ar